Nano-graphene oxide improved the antibacterial property of antisense yycG RNA on Staphylococcus aureus

The antisense yycG sequences (ASyycG) were synthesized by Sangon Biotech (Shanghai, China). To generate a recombinant pDL278 ASyycG plasmid, the ASyycG sequences were inserted into the BamHI and EcoRI restriction sites of a pDL278 vector [16]. To synthesize the GO-PEI complexes, GO powder (XFNANO Materials Tech, Nanjing, China) was added to ddH₂O to a final concentration of 0.1 mg/mL. Next, the solution was slowly mixed with branched polyethyleneimine (BPEI, 10 kDa; Sigma-Aldrich, St. Louis, MO, USA). Then, the solution was processed with 10 cycles of ultrasonication for 60 s, with 60 s rest on ice between each sonication. The obtained solution was mixed on a shaking table overnight at room temperature. To remove redundant PEI compounds, the mixtures were washed three times with ddH₂O by centrifugation (12000×g, 1 min) and resuspended with ddH₂O to a final concentration of 0.1 mg/mL. pDL278 ASyycG plasmid (100ng/μL) was added to the GO-PEI complexes at a volume ratio of 1:125 and the mixtures were incubated for 1 h at room temperature.

The working concentration of GO-PEI-ASyycG was determined by cytotoxicity assays. Briefly, the mouse embryonic fibroblast NIH/3T3 cell line (Sigma-Aldrich) at a density of 1000 cells/well were seeded into 96-well plates in 100 μL of Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), and mixed with the GO-PEI-ASyycG solutions at dilutions ranging from 100 μg/mL to 0 μg/mL. After incubation for 48 or 72 h (37 °C, 5% CO₂), the plates were removed and each well was washed with phosphate buffer solution (PBS, pH = 7.4) twice. The CCK-8 cell counting kit (Dojindo Laboratories, Kumamoto, Japan) was used to test the cell viability and each well was incubated with 10 μL of CCK-8 reagent. After 2 h of culture, the OD values of each well were measured using a microplate reader (ELX800, Gene, Hong Kong, China) at 540 nm.

The particle size distribution of the GO, GO-PEI, and GO-PEI-ASyycG solutions (0.1 mg/mL) was measured by dynamic light scattering (DLS) and the zeta-potential was examined by a Malvern instrument (Zetasizer Malvern Nano ZS, Instruments, Worcestershire, UK). A total of 50 μL of GO, GO-PEI, or GO-PEI-ASyycG solution was dropped onto sterile coverslips and films were prepared and air-dried in room temperature. The roughness of the films was assessed using an atomic force microscope (AFM) (SPM-9500J2, Shimadzu, Tokyo, Japan) in the contact mode. Micrographs of all films were evaluated by scanning electron microscopy (SEM; Inspect F50, FEI, Hillsboro, OR, USA) as previously described [17].

A single colony of S. aureus was selected from a tryptic soy agar (TSA) plate and cultured in tryptic soy broth (TSB) medium to the mid-exponential phase, which was determined by an OD₆₀₀ value of 0.5. For the S. aureus GO group, 250 μL of mid-exponential S. aureus was incubated with 2 μL GO solution (final concentration determined by cell viability assay). In the ASyycG group, 2 μL of recombinant pDL278 ASyycG plasmid was mixed with 250 μL of mid-exponential S. aureus as in our previous studies [16]. For the GO-PEI-ASyycG strains, 250 μL of mid-exponential S. aureus was co-cultured with prepared GO-PEI-ASyycG. All S. aureus strains were cultured at 37 °C in 5% CO₂ for 1 hour, then diluted into 5 mL of fresh TSB medium.

The ASyycG plasmids were labeled with gene encoding enhanced green fluorescent protein (ASyycG-eGFP). The sequences of ASyycG and eGFP were synthesized by Sangon Biotech (Shanghai, China) and are listed in the Additional file 1. The ASyycG-eGFP and GO-PEI-ASyycG-eGFP strains were constructed based on the transformation procedures described above. Both strains were grown in TSB medium until an OD₆₀₀ value of 0.5 was reached. A total of 50 μL of bacterial suspensions was dropped onto coverslips and air-dried at room temperature for 30 min. Confocal laser scanning microscopy (CLSM) was applied to determine the expression level of eGFP. The transfection efficiency was determined by comparing the green fluorescence intensities.

Real-time polymerase chain reaction (RT-PCR) assays were conducted to assess the expression of ASyycG in all S. aureus strains. Briefly, total RNA was extracted from S. aureus suspensions from the mid-logarithmic growth phase in TSB medium using an RNA purification Kit (MasterPure, Epicentre, Madison, WI, USA) according to the manufacturer’s instructions. Total RNA was reverse transcribed using an RT Reagent Kit (PrimeScript, Takara, Kyoto, Japan). Quantitative RT-PCR assays were carried out using the primers listed in Table 1 using a LightCycler 480 system (Roche, Basel, Switzerland). The 16S rRNA gene was used as an internal control [17].

After transformation, all strains were diluted in TSB at a ratio 1:20 and incubated in 96-well plates. The bacterial growth curves were monitored by measuring the OD_600nm with a microplate reader (ELX800, Gene, Hong Kong, China) every 60 min for 24 h. The proportions of live bacteria cells were estimated by confocal laser scanning microscopy (CLSM, FV1000; Olympus Corporation, Tokyo, Japan) at × 40 magnification. Live cells were stained with SYTO9 dye (LIVE/DEAD Bacterial Viability Kit reagent; BacLight, Invitrogen, Grand Island, NY, USA) and dead cells were labeled with propidium iodide (PI). Three-dimensional reconstruction was conducted and analyzed using Imaris 7.0.0 software (Imaris 7.0.0, Bitplane, Zurich, Switzerland) as previously described [18].

Crystal violet (CV) assays were applied to compare the biomass of S. aureus biofilms cultured in 24-well polystyrene plates for 24 h. As previously described, the biofilms were stained with 0.1% (w/v) crystal violet for 15 min at room temperature [17]. The dye bound on the biofilms was collected using 1 mL of de-staining solution (8:2 ethanol: acetone). Then, the solution was transferred to a new plate and the OD_600nm was read by a microplate reader (ELX800, Gene, Hong Kong, China).

Sterile coverslips were immersed in 24-well plates with different S. aureus strain suspensions (OD_600nm = 0.5). After 24 h of co-culturing, the planktonic suspensions were removed and the biofilms grown on the coverslips were washed three times with PBS (pH7.2). Then, the biofilms were fixed in 2.5% glutaraldehyde for 4 h at room temperature and dehydrated with serially concentrated ethanol solutions (30%, 50%, 70%, 80%, 95%, and 100%). The prepared biofilms were dried to critical-point at room temperature and coated with gold powder. Scanning electron microscopy (SEM; Inspect F50, FEI, Hillsboro, OR, USA) was used to estimate the morphologies of all S. aureus biofilms by selecting three random areas from each sample [19].

The viability of the 3T3 fibroblasts cells was significantly decreased after 48 or 72 h treatment GO-PEI-ASyycG concentrations higher than 50 μg/mL (Fig. 1a). Our results indicated that the GO-PEI-ASyycG complexes were not toxic at concentrations lower than 50 μg/mL (Fig. 1a). Dynamic light scattering measurements were applied to determine the hydrodynamic sizes of GO, GO-PEI, and GO-PEI-ASyycG in deionized water. Z-average sizes of 420 nm and 280 nm were obtained for GO and GO-PEI, which were much smaller than the average size of GO-PEI-ASyycG (490 nm) (Fig. 1b). The surface charge (zeta potential) value of GO was approximately at − 23.1 mV. After mixing with the cationic PEI polymer, the GO-PEI and GO-PEI-ASyycG complexes demonstrated positive surface charges of 15.7 mV and 38.4 mV, respectively (Fig. 1c). Using AFM, the roughness analysis of the membranes revealed that the GO-PEI-ASyycG nanosheet roughness averaged 8.9 nm, significantly higher than the GO and GO-PEI films, which averaged 3.4 nm and 4.5 nm, respectively (n = 10, p < 0.05; Fig. 1d). Using SEM, the results showed rougher and denser surface morphologies on the GO-PEI-ASyycG films compared to GO and GO-PEI (Fig. 1e).

Using confocal laser scanning microscopy, higher levels of GFP-expression were observed in samples induced with GO-PEI-ASyycG compared to pure ASyycG (Fig. 2a). A 200% increase in GFP-expression transcripts in GO-PEI-ASyycG transformed strains was found, a statistically significant increase compared to the expression in ASyycG cells (n = 10, p < 0.05; Fig. 2b). Quantitative RT-PCR assays demonstrated that expression levels of ASyycG RNA in the ASyycG and GO-PEI-ASyycG strains were significantly increased 2.8-fold and 6.5-fold, respectively, compared to S. aureus ATCC29213 strains (n = 10, p < 0.05; Fig. 2c). Accordingly, the gene expression levels of yycG were significantly downregulated in the ASyycG and GO-PEI-ASyycG strains (n = 10, p < 0.05). Consequently, the expression levels of PIA synthesis-associated genes icaA/D/B/C were the lowest in the GO-PEI-ASyycG strain among all groups (n = 10, p < 0.05).

When growth was monitored in the different strains, the results showed that the time before entry into the log phase in the GO-PEI-ASyycG strains was obviously prolonged compared to that of the ASyycG strains (Fig. 3a). The most impaired formation of biofilms was observed in GO-PEI-ASyycG strains compared to GO, ASyycG, and ATCC29213 (Fig. 3b). Quantitatively, these results were confirmed by the OD values of the biofilm biomasses, which were the lowest in the GO-PEI-ASyycG strains (n = 10, p < 0.05, Fig. 3b).

SEM observation demonstrated reduced levels of extracellular matrix components in the biofilms of the GO, ASyycG, and GO-PEI-ASyycG cells that were separated by blank areas (Fig. 3c). Exopolysaccharide-enmeshed cell clusters were greatly decreased in the GO-PEI-ASyycG strain biofilms (Fig. 3c). Using CLSM, we demonstrated that the proportion of viable bacteria was significantly decreased in the GO, ASyycG, and GO-PEI-ASyycG strains compared to the S. aureus ATCC29213 strain (Fig. 4a). The lowest percentage of live bacteria was observed in the GO-PEI-ASyycG strain, at 14.83 ± 0.5 % (n = 10, p < 0.05, Fig. 4b).