Synthesis of nano silver
Silver nanoparticles (AgNPs) with similar nano size but opposite surface charges were synthesized based on previously reported methods.
Negative charged AgNPs: based on Vladimir’s method with slight modification , the following freshly prepared solutions were mix together in the following order: 7 ml of 12.5 mM TSC, 17.5 ml of 0.375 mM AgNO3, 17.5 ml of 10 mM hydrogen peroxide (H2O2), 228 ul of 10 mM potassium bromide (KBr). Then 8 ml of 5 mM NaBH4 was added dropwise into the above solution until there was no further visible color change. This brown-red product solution was the AgNPs (named AgNPs-1).
Positively charged AgNPs: based on Abbaszadegan’s method with slight modification , 1 ml of 10 mM AgNO3 was dropped into 20 ml of 6.2 mM 1-dodecyl-3-methylimidazolium chloride ([C12 mim][Cl]) aqueous solution and vigorously stirred. Then 100 ul of freshly prepared 0.4 M NaBH4 was dropped into the resultant solution to obtain a golden solution of AgNPs (named AgNPs-2).
Synthesis of polymer-functionalized AgNPs (P-AgNPs)
PEG, PVP, and Pluronic™ with different weight molecular, including PEG 400, PEG 2000, PEG-SH 5000, PVP (MW 10000), Pluronic P103 (Mn 5000), P123 (Mn 5800), and F127 (Mn 12000), were used to functionalized the synthesized AgNPs. The molar ratio of Ag ion and polymer PEG/PVP was set as 1:4, and the polymers were freshly pre-dissolved in 10 ml of sterile DI water. The Pluronic™ products were directly added to the AgNPs solutions at the value lower than the CMC (critical micelle concentration) value, the CMC value of P103, P123, and F127 were 0.1 mg/ml, 0.052 mg/ml and 1 mg/ml, respectively. Polymer solution/powder of appropriate concentration and weight was added to the corresponding synthetic AgNPs solution to encapsulate the AgNPs, and then the product solutions were stirred overnight for full reaction.
All AgNPs solutions were centrifuged and washed three times at 15000 rpm for 15 min each to remove the excess unreacted ionic liquid. The pellet was then resuspended in 200 ul of sterile DI water and the AgNPs-1/2 and P-AgNPs-1/2 products were stored at room temperature.
The physicochemical properties of AgNPs
UV-Visible (UV-Vis) absorption. The 100x diluted solutions of AgNPs in sterile DI water were prepared to detect the UV-Vis absorption spectroscopy (200 nm-700 nm, 10 nm interval.).
The silver concentration of AgNPs was determined by ICP-OES (inductively coupled plasma optical emission spectroscopy). Briefly, 1 ul of AgNPs was added to 9 ul of 68% concentrated nitric acid to convert silver element into silver ion. Subsequently, 3.99 ml of 1M nitric acid solution was used to dilute the silver ion solution. Thus, 4000-fold diluted solutions of AgNPs were analyzed using ICP-OES.
A series of 10x-100x diluted solutions of AgNPs in sterile DI water (ca. 20 µg/ml) were prepared for the determination of hydrated nano size and zeta potential of AgNPs using dynamic light scattering (DLS) (Malvern, Nano-zetasizer).
The morphology and nano size of AgNPs were detected by Transmission electron microscopy (TEM, Philips CM100). Ten microliters of 10x-100x diluted solutions of AgNPs in sterile DI water were deposited onto 400-mid formvor/carbon-coated copper grid. The samples were put on filter paper and dried at room temperature for 1 h, and then images of the AgNPs were captured using TEM.
The minimum inhibitory concentration (MIC) for AgNPs against planktonic P. gingivilis was determined using the microdilution method based on the Clinical Laboratory Standards Institute (CLSI) guideline . The maximum test concentration of AgNPs for MIC determination was set to be 2% of the volume of the bacterial suspension. In brief, 100 ul of P. gingivilis broth containing serially diluted AgNPs were pipetted into a 96-well cell plate. Subsequently, 108 CFU/ml (OD660nm = 0.271–0.279) of P. gingivilis in the late logarithmic phase was prepared and inoculated into fresh P. gingivilis broth (ratio = 2:100). Then 100 ul of the prepared P. gingivilis bacterial suspension (106 CFU/ml) was pipetted into the 96-well cell plate and inoculated in an anaerobic incubator at 37℃ for 3 days. Absorbance values at OD660nm were detected to assess the growth of P. gingivilis. Negative control and positive controls, including silver diamine fluoride (SDF), AgNO3, and metronidazole (MNZ), were set up parallelly, and three parallel samples and three independent replicates were set up to determine the MIC of AgNPs. The MIC was defined as the lowest concentration that substantially inhibited bacteria growth in the medium.
Time-dependent antibacterial effect of AgNPs
In 96-well cell plate, 200 ul/well of P. gingivilis bacterial suspensions (106 CFU/ml) were prepared and then shocked by AgNPs at MIC level in an anaerobic incubator at 37℃ for 30 min, 2 h, 6 h, and 24 h. Five microliters of suspension was dropped on blood agar and incubated for 5 days. Three independent assays were performed parallelly. The pictures of blood agar were taken with a camera.
Antibiofilm effect of AgNPs on P. gingivilis biofilm
The mature P. gingivilis biofilms were incubated in iBidi Cell Plate which was specifically applied for confocal laser scanning microscopy (CLSM). Briefly, 200 ul of P. gingivilis suspension (108 CFU/ml) in fresh P. gingivilis broth was placed in iBidi Cell Plate and incubated in an anaerobic incubator at 37℃ for 6 days (P. gingivilis broth was refreshed every 3 days). When the mature P. gingivilis biofilms were constructed, we gently discarded the supernatant in the iBidi cell plate and pipetted 200 ul of fresh P. gingivilis broth containing AgNPs at MIC and 10*MIC concentration into cells. After an extra 24 h incubation in anaerobic incubator, we removed the supernatant, washed the cells with PBS and stained the biofilm with SYTO9 and PI dyes (Live/Dead bacterial viability kit, ThermoFisher) for 30 min. The fluorescence images were captured by the CLSM.
In addition, the dose-dependent bactericidal effect of AgNPs and P-AgNPs on mature P. gingivilis biofilm was detected using CCK8 assay. The mature P. gingivilis biofilms were prepared and incubated in 96-well plate. In short, 100 ul of 10% FBS per well was pre-cultured in a sterile 96-well plate for 1 h, 80 rpm at 37℃. After removing the FBS, we added 100 ul of fresh P. gingivilis broth containing 1% of P. gingivilis suspension (108 CFU/ml), incubated the plate in an anaerobic incubator for 6 days at 37℃, and changed the medium every 3 days. Subsequently, we replaced the suspension with fresh medium and performed turbidity measurements at OD600nm (initial value) to confirm the consistency of mature biofilm. Sterile medium containing AgNPs-1/2 and P-AgNPs-1/2 with MIC, 2.5*MIC, 5*MIC, 7.5*MIC and 10*MIC concentrations were freshly prepared at the same time, and were pipetted into the above well plate. After incubation for another 24 hours, the suspension was replaced by fresh medium containing 10% CCK8 and the optical density at 450 nm of well plate were detected in 2 hours. Subsequently, the biofilm was resuspended in 100 ul of fresh medium and incubated on blood agar. CFU below 10 was considered to have no live bacteria.
Cytotoxicity of AgNPs
Human gingival fibroblasts (HGF-1, P6) were used to detect the cytotoxicity of AgNPs. HGF-1 (P3) were resuscitated and passaged in Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. Subsequently, 5000 cell/well (P6) were seeded in a 96-well cell plate. After incubation for an additional 24 h for cell attachment, cells were shocked with fresh medium containing MIC concentration of AgNPs for 6 h, 24 h, and 48 h. The medium was then removed and replaced with 90 ul of PrestoBlue Cell viability reagent (10% in PBS, Thermo Fisher.) for 10 min at 37℃. The fluorescence in 560/590nm (excitation/emission) were read by SpectraMax M2 (USA). Five parallel samples and three independent assays were performed.
Unless otherwise specified, all the graphs were drawn with GraphPad Prism 5 software. All the data were statistically analyzed using the software SPSS V21.0 (IBM Corporation, Armonk, NY, USA). Normality of data distribution was assessed by Shapiro-Wilk test. Differences between different groups were assessed by t-test and one-way ANOVA with Bonferroni multiple comparison tests. A p-value at or less than 0.05 is statistically significant.