Self‐Assembled Soft Nanomaterials Via Silver(I)‐Coordination: Nanotube, Nanofiber, and Remarkably Enhanced Antibacterial Effect

Silver(I)‐induced instant gelation of pyridine‐containing Fmoc‐l‐glutamate and its concentration‐dependent self‐assembly from nanotubes to nanofibers are investigated. The formed metallogel with nanostructure has remarkably enhanced antibacterial activities. Interestingly, the nanotube and nanofiber exhibit different antibacterial activities, and a corresponding antimicrobial mechanism is proposed.

Self-assembled soft nanomaterials via various noncovalent bonds have been attracting great interest due to their easy of fabrication and tailor-made functions. [ 1 ] Among various soft nanomaterials, metallogels [ 2 ] that incorporate the metal ions into the supramolecular gel endowed the gel with certain unique features including the redox, mechano, magnetism, pH responsiveness, [ 3 ] and new application potentials such as effective electron-and light-emitting nanomaterials, asymmetric catalysis, visual chiral recognition, and chemosensors. [ 4 ] While many of these functions were extensively investigated, the bioeffects of the gel materials were less reported. [ 5 ] Among various metals, silver and its compounds (including silver nanomaterials) are historically well known and extensively investigated as antimicrobial agents to fi ght infections and control spoilage. [ 6 ] Since the increasing of antibiotic-resistant bacterial strains for the conventional antimicrobial treatments, free metal ions such as silver ions in large quantities are used, especially to fi ght against antibiotic-resistant pathogens. [ 7 ] Nowadays, a variety of silver-containing biomedically relevant materials are exploited and used in clinical treatments including dental work, catheters, and burn wounds. [ 8 ] It is expected to develop new materials with higher effi ciency and less silver usage. The design and utility of silver-coordinated metallogels, which might be able to show good stability, biocompatibility, and sustained effective antibacterial activity, come to our sight. [ 9 ] Unfortunately, the antibacterial activity of these metallogels related to the microscopic nanostructures and the acting antibacterial mechanism have still not been completely understood. Herein, combining the structural features of metallogel and the good antimicrobial activity of silver(I)-pyridyl coordination compounds, we report the silver(I) metallogels with tunable assembly structures from wileyonlinelibrary.com metallogels exhibited typical solid-like rheological behavior with G ′ an order of magnitude larger than G ″ in the frequency range of 1-100 rad s −1 . Interestingly, the storage modulus G ′ and the loss modulus G ″ greatly increased with the gel concentration, which suggested that the higher gel concentration, the much better mechanical rigidity. On the other hand, we further measured the sol-gel transition temperature ( T gel ) to characterize the thermal stability of these metallogels. T gel also showed a concentration dependency, as shown in Figure S2B (Supporting Information). The T gel obviously increased from 51 to 78 °C with the gel concentration from 4 to 16 × 10 −3 M .
The concentration-dependent fl uorescence spectra starting from very dilute solution to the assembling gel state have been studied to disclose the aggregation behavior of fl uorene moieties within these metallogels ( Figure 2 A). In the case of dilute solution, the emission peak of the fl uorenyl moieties around www.MaterialsViews.com www.advancedscience.com Scheme 1. A) Molecular structure of 4MPFG. B) Schematic illustration of silver ion-induced instant gelation. The self-assembly behavior of 4MPFG is highly dependent on the concentration of gelators and the self-assembled nanotube and nanofi ber structure can be obtained at different concentration. Thus, ring-shaped and helical chain-type coordination mode is proposed. The formed nanomaterials show differential antibacterial activity when they are utilized as the antibiotic agents. The antimicrobial mechanism is that the nanomaterials can destroy the membrane integrity and induce DNA condensation to fi nally kill the bacteria. (3 of 7) 1500134 wileyonlinelibrary.com 315 nm with the shoulder peak at 306 nm was the characteristic peak of the fl uorenyl monomer. Fluorescence intensity slowly increases by increasing the concentration from 0.5 to 8 × 10 −3 M . This is due to the enhancement of concentration of gelator molecules. Meanwhile, gradual redshift of the fl uorescence emission maximum from 315 to 323 nm was observed, during the gelator concentration increased to 8 × 10 −3 M , which suggest that the two fl uorenyl moieties overlap in parallel fashion. Further enhancement of gelator concentration from 8 to 16 × 10 −3 M induced the emission peak continuously shifting to the long wavelength and fi nally located at 330 nm, which indicate the antiparallel packing of fl uorenyl moieties. [ 10 ] 1 H NMR spectra of 4MPFG and 4MPFG with Ag + at the concentration of 8 and 16 × 10 −3 M were measured in DMSO -d 6 solvent (Figure 2 B). With the increase of concentration, the 2,7 and 3,6-H signals of the fl uorenyl moieties were gradually shifted downfi eld and 1,8 and 4,5-H exhibited slightly upfi eld shift, which is the indication of antiparallel stacking of fl uorenyl moiety. [ 11 ] Moreover, the proton signals of pyridine ring showed downfi eld shift when Ag + was added, which is due to the coordination between the pyridine and Ag + imparting the electronwithdrawing inductive effect on the proximate protons. [ 12 ] The supramolecular arrangement in Gel 1 and Gel 2 was further measured by circular dichroism (CD) spectroscopy since the gelator has a chiral center (Figure 2 C). As expected, no obvious CD signal was detected in the solution of 4MPFG. However, the supramolecular chirality transfer from L -glutamic acid to achiral fl uorenyl groups and pyridine rings was detected in both metallogels. The positive Cotton effect at 228 nm and negative at 209 nm in Gel 1 suggest the helical arrangement of amide groups. [ 13 ] Meanwhile, the positive bands from 255 nm and the negative Cotton effect with a double minima at around 305 and 273 nm were observed, which indicate the superhelical packing of the pyridine and fl uorenyl moieties, respectively. [ 10 ] Similar CD signals of amide and fl uorenyl moieties were obtained in Gel 2, but the intensity was obviously strengthened, which suggest that those parts of groups were packed more closely in Gel 2. Moreover, the positive CD band of pyridine moieties was shifted to 251, which may be due to the different packing mode of pyridine rings.
In order to further investigate the structure of assemblies, small-angle X-ray diffraction (XRD) spectra of the two xerogels were measured, as shown in Figure 2 D. The well-developed diffraction patterns for the assemblies and d -spacing values were estimated based on the Bragg's equation. The lamellar packed structure with a layer distance of 4.4 nm was obtained in Gel 1. This d -spacing value is longer than the calculated molecular length (as simulated ≈1.8 nm) and roughly equals the length of two 4MPFG molecules with coordinated silver ions. In the case of Gel 2, the diffraction patterns were observed at d -spacing value of 4.4, 3, and 2.3 nm estimated in the ratio of 1: 1/2 : 1/4 , which is in good agreement with columnar square phase packing. [ 14 ] Fromm et al. have previously reported a phenomenon about the concomitant crystallization of two polymorphs. Two supramolecular isomers, a ring and a helix, are isolated from the same mother liquor as a result of concentration effects. [ 15 ] Based on their work and the above results, two different self-assembly www.MaterialsViews.com www.advancedscience.com Figure 2. A) Concentration-dependent fl uorescence spectra of 4MPFG/Ag + metallogels starting from very dilute solution (concentration 0.5 × 10 −3 M , λ ex = 265 nm) to different gel state (concentration from 1 to 16 × 10 −3 M , λ ex = 265 nm); B) 1 H NMR spectra of 4MPFG, Gel 1 and Gel 2 in DMSOd 6 ; C) CD; and D) XRD spectra of Gel 1 and Gel 2.
wileyonlinelibrary.com mechanisms due to the concentration effect can be proposed as illustrated in Scheme 1 . In Gel 1, the relative concentration of 4MPFG is low and silver ions tend to coordinate with the minor gelators. Therefore, an oval-shaped ring that the two 4MPFG molecules are bridged by two silver ions is apt to be formed. Since the π-π stacking of fl uorenyl moieties and the hydrogen bonding interactions as the main driving force in orthogonal direction, the oval-shaped ring adopted face-to-face packing and then crimped to form nanotube structures. On the other hand, in Gel 2, the concentration of 4MPFG is higher with respect to the Gel 1 and the major gelators will be linked by silver ions to form the helical coordination polymer chains. These coordination polymer chains benefi ted the antiparallel stacking of the fl uorenyl groups and fi nally closely packed according to columnar square phase to form the twisted nanofi bers.
To determine whether the biological effects of the metallogels are closely correlated to their structures, we evaluated the antibacterial activities of the synthesized xerogels against Gram-positive bacteria S. aureus and S. epidermidis , and Gramnegative bacteria Escherichia coli ( E. coli ) using broth inhibition assay (details are shown in the Experimental Section). As shown in Figure 3 A and Figure S3 (Supporting Information), with the equimolar amount of Ag(Ι) ion, the Gel 1 and Gel 2 show better bacteriostatic activities than AgNO 3 or the mixture of AgNO 3 and ligand to S. epidermidis , E. coli , and S. aureus . However, no signifi cant change of antibacterial activity is observed between AgNO 3 and the mixture of AgNO 3 and ligand, which suggesting that the formation of nanostructure played a key role for the inhibition of bacteria growth. More interestingly, different antibacterial results to S. epidermidis and E. coli are obtained between Gel 1 and Gel 2. This may be due to the different self-assembled nanostructure between Gel 1 and Gel 2. Silver sulfadiazine (SD-Ag) has received widespread acceptance as a topical agent to control bacterial infection, especially in helping heal burn wounds. [ 16 ] Therefore, herein we compared the effect of SD-Ag and metallogels on the growth curve of S. epidermidis , E. coli , and S. aureus . To our surprise, the metallogels exhibit better antibacterial activities than SD-Ag against all bacteria ( Figure S4, Supporting Information). Moreover, Gel 2 has better antibacterial activities than Gel 1 to S. epidermidis www.MaterialsViews.com www.advancedscience.com and E. coli , which is in agreement with the broth inhibition assay. Together, these results suggest that the nanostructure of metallogel plays the critical role in their antibacterial activity.
To further explore the antibacterial mechanisms of metallogel, we used environmental scanning electronic microscopy (ESEM) and TEM to investigate the detailed ultrastructural changes caused by metallogels within the cell, such as the loss of membrane integrity, DNA condensation, and cytoplasmic reorganization. As shown in Figure 3 B and Figure S5 (Supporting Information), signifi cant morphological changes occurred in S. epidermidis , E. coli , and S. aureus cells after the addition of Gel 1 or Gel 2. The loss of membrane integrity can be clearly observed via ESEM and TEM in S. epidermidis and E. coli cells exposed to low-dose Gel 1 and Gel 2. Moreover, there are intracellular substances released from some of the S. epidermidis and E. coli cells treated with high-dose Gel 1 ( Figure S6, Supporting Information). Importantly, the leakage of intracellular substances can be more obviously observed in cells treated with Gel 2, which provides one explanation for why Gel 2 has better antibacterial activities than Gel 1.
As examined by TEM, the untreated S. epidermidis and E. coli cells show unanimous electron density, and the DNA molecules are distributed randomly in almost all parts of the cells, suggesting that the cells are in a normal condition without environment disturbance. [ 17 ] However, after treatment with Gel 1 or Gel 2, the cytoplasm membrane shrank or detached from the cell wall in S. epidermidis and E. coli cells. Moreover, there are many condensed DNA molecules positioned in the center of cells, which are indicated by white arrows. The replication of DNA molecules is effectively conducted only when DNA molecules are in a relaxed state. In a condensed form, DNA molecules lose their replicating abilities, thus the cytokinesis of cells will be blocked. [ 18 ] In accord with these fi ndings, there is a large increase of cytokinesis-blocked S. epidermidis cells (indicated by white arrows) observed by both ESEM and TEM after the treatment of both Gel 1 and Gel 2. In addition, some S. epidermidis cells cannot fi nish their cell mitosis before they go to death. Therefore, metallogel-caused DNA damage may be responsible for their antibacterial activities.
To further illustrate correlation between metallogel-caused membrane disruption and the antibacterial activities of metallogels, we investigated the effect of metallogels on the permeability of cell membranes using the propidium iodide (PI) staining method. PI can intercalate within DNA and RNA to form a bright red fl uorescent complex, but it cannot cross the membrane when the cell is alive. Therefore, intracellular staining of PI can specifi cally identify permeable cells. As shown in Figure 4 and Figure S7 (Supporting Information), both Gel 1 and Gel 2 induce serious permeability of cell membranes and leakage of nucleic acids in all bacteria. With an equimolar amount of Ag, the Gel 1 and Gel 2 induce more serious permeability of cell membranes and leakage of nucleic acids in all bacteria than the mixture of AgNO 3 and ligand. Moreover, at a dose of 20 × 10 −6 M Ag in Gel 1 and Gel 2, the percentage of permeable S. epidermidis with Gel 2 is nearly onefold higher than that with Gel 1; and the percentage of permeable E. coli with Gel 2 is also higher than that with Gel 1. These fi ndings suggest that Gel 2 cause more damage to cell membrane of S. epidermidis and E. coli than Gel 1, which may be one reason why Gel 2 has better antibacterial activities than Gel 1 against S. epidermidis and E. coli .
In summary, a bispyridyl-conjugated Fmoc-L -glutamate is found to form instant gel at room temperature as soon as the incorporation of silver salt. The instant metallogel exhibits thermally reversible sol-gel transition and its self-assembled behavior is largely dependent on the gelator concentration which result in distinct structural change from nanotube to nanofi ber. The corresponding xerogels show good inhibitory activity against the growth of Gram-positive and Gram-negative bacteria and exhibit great potential to be utilized as better antibacterial reagents than SD-Ag. Moreover, the metallogels with the two different self-assembled nanostructures showed tunable antibacterial activities. Nanofi bers may cause more damage to cell membrane than nanotubes and thus showed better antibacterial activities. The antibacterial results suggest that these 4MPFG/Ag + metallogels may have valuable applications in various fi elds, such as the manufacture of household appliances and medical devices.

Experimental Section
Synthesis of 4MPFG : All the starting reagents were purchased from commercial suppliers and used without further purifi ed. N , N ′-bis(pyridin-4ylmethyl)-Fmoc-L -glutamate was synthesized by the amidation of Fmoc-L -glutamic acid with 4-(aminomethyl)pyridine according to the following method. The compound Fmoc-L -glutamic acid (3.69 g, 0.01 mol) and 4-(aminomethyl)pyridine (2.03 mL, 0.02 mol) with a catalytic amount of 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC·HCl, 5.75 g, 0.03 mol) and 1-hydroxybenzotrizole (HOBt, 4.05 g, 0.03 mol) were mixed in dry CH 2 Cl 2 (100 mL, 250 mL fl ask) and the reaction mixture was stirred for 72 h at room temperature. After the reaction, the solvent was removed by rotary evaporation. The resultant mixture was dissolved in 20 mL ethanol by heat and poured into 500 mL pure water. The precipitation was fi ltered and the crude product was obtained. After purifi cation by silica column chromatography (CH 2 Cl 2 / CH 3 OH = 10/1, R f = 0.5), the target product was obtained as a white solid (4.82 g, 88% yield). 1 H 5.69, N 12.74; found: C 69.99, H 5.97, N 12.51.
Apparatus and Measurements : 1 H NMR spectra were recorded on a Bruker AV400 (400 MHz) spectrometer. Mass spectral data were obtained by using a BIFLEIII MALDI-TOF MS instrument. Elemental analysis was performed on a Carlo-Erba-1106 Thermo-Quest. CD spectra were obtained using JASCO J-810 CD spectrophotometers. Rheological studies were achieved on a Discovery DHR-1 rheometer (TA Instruments). The rheology experiments were performed at 25 °C using parallel plate geometry in a Peltier plate (40 mm diameter aluminum plates). Fluorescence spectra were measured on an F-4600 fl uorescence spectrophotometer using a xenon lamp as the excitation source. X-ray diffraction (XRD) was achieved on a Rigaku D/Max-2500 X-ray diffractometer (Japan) with CuKα radiation ( λ = 1.5406 Å), which was operated at 45 kV, 100 mA. SEM measurements were performed on a Hitachi S-4800 FE-SEM microscope. TEM images were obtained on a JEM-1011 electron microscope operating at accelerating voltages of 200 kV. The fl uorescence of PI staining was excited by a 559 nm laser and observed with a laser scanning confocal microscope (Olympus, FV1000-IX81). The morphology of bacterial was characterized by Metallogels Fabrication and Characterizations : A series of ethanol solution of 4MPFG and AgNO 3 aqueous solution with the concentration from 1 to 32 × 10 −3 M were prepared. 0.5 mL 4MPFG solution was fi rst added in a capped test tube. Then 0.5 mL AgNO 3 aqueous solution with the corresponding concentration was added into the above solution. The metallogels were instantly formed and incubated at 60 °C for 5 min under the darkness. The sealed test tube was then allowed to cool down to the room temperature. The formed metallogels were then washed by pure water for three times to remove the uncoordinated silver ions and separated by centrifuge at 10 000 rpm and fi nally dried under vacuum for 24 h to obtain the corresponding xerogels. For the TEM and SEM measurements of gel morphology, a small amount of dilute metallogels were placed onto a carbon-coated copper grid (unstained) or a singlecrystal silicon plate (Pt coated), respectively, after being vacuum dried for 12 h. In the case of preparing samples for XRD measurements, gels were cast onto glass plates and dried under vacuum. In the process of measuring the CD and fl uorescence spectra of metallogels, a quartz cuvette with 0.1 mm width was used.
Determination of Antibacterial Activities of Metallogels : In the broth inhibition assay, bacteria were cultured in the nutrient broth medium (5 g L −1 NaCl, 10 g L −1 tryptone powder, and 5 g L −1 beef extract powder, pH = 7.2) at 37 °C on a shaker bed at 200 rpm for 8-10 h and diluted with the broth to an optical density of 0.3 at 600 nm measured with UV-vis spectroscopy (Varian-Cary Bio100), then we added 200 µL of each aqueous solution of Gel 1, Gel 2, AgNO 3 , AgNO 3 + 4MPFG, or water into 4.8 mL of diluted broth containing bacteria in test tubes and cultured them at 37 °C on a shaker bed at 200 rpm for another 12 h. Finally, 2 mL of each mixture after incubation were transferred into a cuvette, and the OD was read with UV-vis spectroscopy at 600 nm against a reagent blank treated in the same manner. For the growth curve experiments, bacteria were cultured as above and diluted to an optical density of 0.15 at 600 nm, and then 10 µL of each aqueous solution of Gel 1, Gel 2, SD-Ag, or water were added into 250 µL of diluted broth containing bacteria in Corning 96-well plate. OD 600 nm was examined at different time courses using Tecan infi nite 200 multimode microplate readers. Cultures were prepared in triplicate, and all experiments were repeated twice or more.
Examination of Permeability of Cell Membranes by Fluorescence Assay : Bacteria suspension (with a 0.3 optical density at 600 nm) mixed with Gel 1, Gel 2, AgNO 3 + 4MPFG at a fi nal concentration of 20 or 40 × 10 −6 M was cultured at 37 °C for 12 h on a shaker bed at 200 rpm, the suspension was collected by centrifugation at 8000 rpm for 3 min, and washed with phosphate-buffered saline (PBS, 0.01 M , pH = 7.4) twice. These bacteria were used as fl uorescence assay, ESEM and TEM For each group of image, the left half shows an image in the differential interference contrast mode, while the right half shows the corresponding fl uorescence image. B, C) S. epidermidis , S. aureus , and E. coli were treated with the indicated dose of Gel 1 or Gel 2, and then the percentage of cells with permeable membranes was calculated by counting three or four microscope fi elds from three independent experiments (each fi eld includes 50-100 cells).
(7 of 7) 1500134 wileyonlinelibrary.com www.MaterialsViews.com www.advancedscience.com samples. The bacterial suspensions were incubated with an equal volume of the propidium iodide solution (3 × 10 −6 M in PBS) in the dark for 30 min at room temperature, washed with PBS twice, and 20 µL of samples placed on a glass slide with a glass coverslip. The control assay was performed without any treatment. The fl uorescence excited by a 559 nm laser with a laser scanning confocal microscope (Olympus, FV1000-IX81) was observed.
Preparation for ESEM and TEM Samples : Bacteria were prepared as above. For ESEM assay, metallogel-treated bacterial samples were fi xed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH = 7.4) for 24 h, dehydrated with series-grade ethanol, and critical-point dried in CO 2 . Finally, cells were scanned with an ESEM. For TEM assay, metallogeltreated bacterial samples were fi rst fi xed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH = 7.4) for 24 h, washed with PBS twice, further fi xed with 1% OsO 4 in PBS for 1 h, dehydrated in a graded series of ethanol solutions, treated with propylene oxide, and embedded in Durcupan. ≈80 nm thick sections were cut, placed on carbon fi lm supported by copper grids, stained with uranyl acetate and lead citrate, and observed with a biological transmission electron microscope (HT7700) at 80 kV.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.