GO was synthesized following a modified Hummers’ method (Morimoto et al., 2016) and obtained as an aqueous dispersion with a concentration of 3 mg/mL. The solvents were obtained from commercial suppliers and used without purification. Water was purified using a Millipore MilliQ^® filter system equipped with the free endotoxin Polisseur Biopak. When stated, the suspensions were sonicated in a water bath Elmasonic P sonicator with settings at 20 W and 40 kHz. MWCO 12,000-14,000 Da dialysis membranes were purchased from Spectrum Laboratories, Inc. Polymyxin B sulfate salt (mixture of polymyxin B1 and B2) and 2,2′-(ethylenedioxy)bis(ethylamine) were purchased from Sigma Aldrich. Depending on the types of containers employed, either 50 mL conical tubes or 1.5 mL microtubes, two centrifuges (Eppendorf Centrifuge 5804R or Eppendorf Centrifuge 5415R) were used.

Morphological analyses were performed using a transmission electron microscope (Hitachi 7500, Hitachi High Technologies Corporation, Tokyo, Japan). The images were analyzed with FIJI software. TGA was performed on a TGA1 (Mettler Toledo) apparatus from 30°C to 900°C with a ramp of 10°C/min under N₂ using a flow rate of 50 mL/min and platinum pans. For GO materials, samples were lyophilized before analysis. XPS analysis was performed on a Thermo Scientific K X-ray photoelectron spectrometer with a basic chamber pressure of 10^-8-10^-9 bar and an Al anode as the X-ray source (1486 eV). The samples were analyzed as powder pressed onto a scotch tape (3MTM EMI Copper Foil Shielding Tape 118). Spot size of 400 μm was used for analysis. The survey spectra were an average of 10 scans with a pass energy of 200 eV and a step size of 1 eV. For each sample, the analysis was repeated three times. A flood gun was turned on during the analysis. We grouped the functional groups of C1s spectra to avoid imprecision due to the proximity of the peak values and the overlapping of some bounds. Therefore, the C1s spectra were deconvoluted in C=O (287.6-289.9 eV) for carbonyl groups, C-O (286.2-287.2 eV) for hydroxyl and epoxide groups, C-N (285.9-286.2 eV) for the amine group and sulfur-containing groups and C-C (284.4-285.3 eV) for sp² and sp³ carbon atoms. For the C-N peak, a range of error was considered since the binding energy of this bond overlaps with the C-O and C-C areas. For data analysis, CasaXPS (2.3.18) software was used. A Shirley background subtraction was applied. A line shape of 70% Gaussian/30% Lorentzian [GL(30)] was selected for all peaks. The FWHM was constrained to be the same for all peaks, apart from the π-π* peak because it is a broad signal. The N1s spectra were deconvoluted into two main peaks at 399.5 eV (N-R₂) for the amine and 401.5 eV (N⁺-R₃) for the ammonium. Infrared spectra of GO were recorded using an attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) Alpha spectrometer from Bruker, with a diamond crystal as a refractive element, in the range 500-4000 cm^-1 at a resolution of 4 cm^-1.

Two mg of solid GO-TEG-PMB was dispersed into 1 mL of fresh LB medium (sonication bath; Elmasonic P30H,37 kHz). The solution was split into two microcentrifuge tubes (1.5 mL) and kept in the dark at 37°C for 1 day for one of the tubes and 7 days for the other tube. Each microtube was centrifuged at 12000 rpm for 15 min and the supernatant was analyzed by HPLC. HPLC analyses were performed on a Waters Alliance e2695 separations module instrument equipped with an autosampler and 2998 PDA detector, on a Nucleosil C18 column (150×4.6 mm), using a 20 min linear gradient of 0.1% TFA in water to 31% or 100% of 0.08% TFA in acetonitrile at a flow rate of 1.2 mL·min^-1.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of tested agents were determined by microdilution method as described previously (Cai et al., 2015). Briefly, overnight grown cultures of P. aeruginosa and E. coli were appropriately diluted, and 100 μL of diluted bacterial suspensions were mixed with a serial dilution of nanomaterials. The bacterial concentration in the mixtures was 1–2×10⁵ colony-forming units (CFU)/mL, and the concentration of tested agents ranged from 1.56 to 100 μg/mL in a series of twofold dilutions. The samples were incubated at 37°C for 24 h, and optical density at 600 nm (OD₆₀₀) was measured. The MIC was defined as the lowest concentration of tested agent which inhibited bacterial growth. For defining the MBC, 10 μL of mixtures described earlier (bacteria and nanomaterials) were placed on agar plates and incubated at 37°C overnight. MBC values were defined as the lowest concentration of the tested agent which prevented the growth of bacteria on agar plates. The treatment time dependent bactericidal efficiency of GO, GO-TEG, and PMB complexed to the different GO was tested against the planktonic cells of P. aeruginosa and E. coli. To prepare a bacterial inoculum, single colonies of P. aeruginosa and E. coli were inoculated to 5 mL of LB medium and incubated overnight at 37°C with continuous agitation. For this assay, 20 μL of inoculum (2–5×10⁷ CFU/mL bacterial cells) from overnight grown bacterial cultures were inoculated into fresh LB medium containing sterile deionized water (control), MIC, and 2 times MIC concentration of tested agents. All samples were incubated at 37°C with continuous agitation for 24 h. A fraction of culture (100 μL) from each sample was taken to determine bacterial viability at time points of 0, 4, 8, and 24 h. The collected samples were serially diluted in 0.89% of a saline buffer. From the diluted samples, 100 μL were plated onto LB agar plates, incubated at 37°C for 24 h, and the number of colonies was counted.

Biofilms of E. coli and P. aeruginosa were formed on 15 mm cover glass. Briefly, overnight grown bacterial culture was diluted with fresh LB to make final inoculum 2×10⁶ –5×10⁶ CFU/mL. 300 μL of bacterial inoculum were loaded to cover glass and incubated at 37°C without any disruption to achieve biofilm formation. After 24 h, the culture medium was replaced with fresh LB medium (control) or MIC and 2×MIC of tested agents, and the samples were further incubated for 24 h. After 24 h of treatment, biofilms were collected in 5 mL of 0.89% of NaCl and homogenized by sonication. The homogenized biofilm suspensions (100 μL) were serially diluted and plated on LB agar plates and incubated overnight at 37°C. The number of colonies was counted to determine the viability of bacterial cells (CFUs). To visualize the live and dead cells, control biofilms and biofilms treated with test agents were stained for 20 min with a mixture of 6.0 μM SYTO 9 and 30 μM propidium iodide from a live/dead BacLight Viability kit L13152 (Thermo Fisher Scientific). Fluorescence microscopic imaging of the biofilms was performed using a Zeiss fluorescence microscope (Axio Imager.Z2m; Carl Zeiss Meditec AG, Jena, Germany). Scanning electron microscopy (SEM) analysis of biofilms was performed as described previously (Pandit et al., 2020). Briefly, biofilms were fixed with 3% of glutaraldehyde for 2 h and dehydrated with graded series of ethanol concentrations (40%, 50%, 60%, 70%, 80%, and 90% for 15 min each) and with absolute ethanol for 20 min. The dehydrated biofilm samples were dried at room temperature overnight and coated with gold before SEM imaging, performed with a Supra 60 VP microscope (Carl Zeiss AG, Jena, Germany).

In order to deploy functionalized GO-PMB as a surface coating, a commercially available catheter was purchased, sliced into pieces (12 mm) and sonicated for 2 h with the sterile water containing 100 µg/mL of GO and GO-TEG, 2×MIC concentration of PMB complexed to GO and GO-TEG (Sonication bath, 100% of amplitude). The coated catheter tubes were dried, and UV sterilized before testing the biofilm formation. The inoculum for biofilm formation was prepared by using overnight grown culture of P. aeruginosa and E. coli and it was diluted with fresh LB to make the final inoculum 2×10⁶ –5×10⁶ CFU/mL. The coated and non-coated catheter tubes were individually placed in culture tube containing 2 mL of inoculum. The culture tubes were incubated at 37°C for 24 h. After 24 h, biofilm on catheter tube was dip washed using sterile water and fixed with 3% of glutaraldehyde for 2 h. After fixation samples were dehydrated with graded series of ethanol concentrations (40%, 50%, 60%, 70%, 80%, and 90% for 15 min each) and with absolute ethanol for 20 min. The dried samples were sputter coated with gold and analyzed by SEM imaging.

All microbiology data are presented as the mean ± standard deviation from at least three different biological replicates. Intergroup differences were estimated by one-way analysis of variance (ANOVA), followed by a post hoc multiple comparison (Tukey) test to compare the multiple means. Differences between values were considered statistically significant when the P-value was < 0.05.

The starting GO was obtained from a modified protocol of Hummers oxidation of graphite, leading to a stable aqueous dispersion at the concentration of 3 mg/mL. According to TEM images, single layer GO had an average size of 1.49 ± 0.9 µm (Figure S1). The adsorption of PMB on the surface of GO was carried out at room temperature by mixing the two components in aqueous solutions (Figure 1). To achieve a homogenous adsorption, the mixture was exposed to an ultraturrax-generated vortex during the addition of the peptide to GO. The HPLC analysis of collected supernatant from washing process demonstrates the absence of free PMB (Figure S2). The complex was subsequently freeze-dried to maintain its structure and prevent peptide release in the solution during storage. After redispersion of the complex in an aqueous solution, its colloidal stability was reduced compared to GO alone (Figure S3).

To improve the stability of the dispersion, GO was modified by covalent functionalization with a short polyethylene glycol (PEG) chain to promote the dispersion of materials and particles in an aqueous environment. The primary amines of NH₂(CH₂CH₂O)₂CH₂CH₂NH₂ (TEG) were allowed to react with the epoxide rings present onto the surface of GO (Figure 1). This approach of GO functionalization is an easy and practical method to reach a high level of functional groups under mild conditions. Verification and quantification of the amino functions were performed after the purification of GO-TEG by dialysis using the Kaiser test. The concentration of the amines resulted in 442 µmol/g of GO. The order of magnitude is similar to that calculated by XPS (577 µmol/g of GO) (vide infra). Then, PMB was adsorbed onto the surface of GO-TEG under the same condition used for non-functionalized GO. TEG functionalization considerably increased the complex dispersion in an aqueous solution (Figure S3). In addition to HPLC, the different complexes were also characterized by TGA, XPS, and FTIR.

TGA, conducted under an inert atmosphere, allowed us to evaluate the thermal degradation profile of GO conjugates (Figure 2, left). In the temperature range between 30 and 800°C, GO conjugates lost more weight than pristine GO. Calculation on the derivative thermogram (Figure 2, right) revealed that GO exhibits the typical two-step degradation profile, with a first modest step at around 100°C and a second one at around 200°C, associated with the loss of water and labile oxygenated groups, respectively. After the loss of water and labile oxygenated groups, an additional step could be observed between 300-400°C, depending on the type of functionalities added to the material surface. This additional step in the thermal profile can be attributed to polymer degradation for GO-TEG and polypeptide PMB degradation for GO-PMB and GO-TEG-PMB, respectively. In addition, there is also a noticeable shift in the step of degradation around 200°C for materials containing TEG chains (Figure 2, right). This shift is caused by a change in the surface chemistry of GO caused by the covalent functionalization, which opened the epoxide ring and introduced secondary amines (less thermally stable). The different functionalization steps increased the weight of the compounds, which translates into a greater loss of mass observable above 450°C for the different conjugates, following this trend: GO-TEG (+2.2% compared to GO), GO-PMB (+2.8% compared to GO) and GO-TEG-PMB (+3.3% compared to GO-TEG).

Subsequently, XPS was performed to study the surface composition and chemical state of the different GO conjugates before and after the complexation with PMB (Figure 3). The addition of the TEG or PMB resulted in clear changes in the XPS spectra, as expected. In comparison to the pristine GO, which was mostly composed of C and O atoms, the addition of TEG and/or PMB increased the number of N atoms. The N1s spectra of functionalized GO show two peaks, at 399.6 and 401.6 eV, corresponding to amines and positively charged amines, respectively. In the case of the high-resolution C1s peaks of the different conjugates, the contribution of C-O bonds is reduced in comparison to pristine GO, implying that the introduction of TEG chain or the peptide increases the ratio of C-C bonds. Furthermore, in comparison to the initial GO, a novel contribution to the C1s deconvolution peak at 286.0 eV was identified. Even if this contribution overlaps with the value of the C-OH binding energy, the appearance of this one is caused by the formation of the bond C-N on the surface during the opening of the epoxides by the primary amine of the chain TEG, and by the presence of the PMB, which contains many amide bonds and the unnatural amino acid 2,4-diaminobutyric acid. The analysis of the XPS surveys allowed us to calculate the number of functional groups. The initial content of nitrogen on GO was 0.9%, and the addition of PMB and TEG considerably augmented this amount (Table at the bottom of Figure 4). Considering the molecular weight of each function, the number of N atoms in the molecules, and the value obtained from the starting GO, the loading of TEG was calculated to be 577 µmol/g of GO (8.5 wt%) for GO-TEG. The loading of PMB corresponded to 281 µmol/g of GO (39 wt%) for GO-PMB, and 228 µmol/g of GO (32 wt%) for GO-TEG-PMB.

Finally, we analyzed the FT-IR spectra of the different GO conjugates to confirm the covalent functionalization with TEG as well as the coupling with PMB (Figure S4). In the spectrum of the starting GO, the broad peak around 3400 cm^-1 was assigned to the O-H stretching of the adsorbed water and the hydroxyl functions of GO. The band at 1723 cm^-1 in GO was attributed to the stretching of C=O groups, while the peak at 1619 cm^-1 corresponded to the H-O-H bending vibration of water molecules and the skeletal C=C bond vibrations of the graphitic domains. The band at 1371 cm^-1 could be assigned to the O-H bending vibration. The C-O-C vibration band of the epoxides was located at 1232 cm^-1 and the peak at 1143 cm^-1 was assigned to the C-O stretching. The presence of amide I and amide II bands in conjugated GO-PMB and GO-TEG-PMB supports the existence of adsorbed PMB onto the surface of GO. The two peaks located at 1647 cm^-1 and 1533 cm^-1 in free PMB can also be found in GO-PMB and GO-TEG-PMB.

Our choice of non-covalent complexation between GO and PMB can raise the issue of uncontrolled release of the peptide during the dispersion in the cell culture media and incubation with bacteria. In this context, we investigated the stability of our GO-TEG-PMB conjugate over time. The material was dispersed in LB culture media using a sonication bath, to prepare two dispersions of 500 µL at 2 mg/mL of GO-TEG-PMB. The dispersions were placed in the incubator at 37°C. The first mixture was centrifuged after 1 day and the second one after 7 days. The supernatants were collected and analyzed by HPLC (Figure 4A). A calibration curve with PMB at different concentrations in LB medium was performed (Figure 4B) to calculate the quantity of PMB released over time (Figure 4C). The chromatogram peaks between 10 and 12 min correspond to PMB identified in LB medium. The first belongs to PMB B2 and the second to PMB B1. The two compositions of PMB differ by the terminal fatty acid chain, with PMB B2 possessing a methyl group, while in PMB B1 the analogue chain is an ethyl group. The presence of PMB B2 in the medium is also visible in the chromatograms of the two supernatants at day 1 and day 7. The analogue with the longer fatty acid chain (B1) seems to remain adsorbed more strongly onto the surface of GO than PMB B2, which is mainly released after 24 h. The calculations allowed to establish a release of 98 µg per 1 mg of GO-TEG-PMB after 24 h and 112 µg after 7 days, corresponding to 10% and 11% of release, respectively. On the basis of these data, we can conclude that almost all PMB B2 is rapidly detached from the surface of GO by comparing the ratios of PMB B1 and PMB B2 (3:1), as well as the previously estimated loading (0.32 g of PMB for 1 g of conjugate).

The antimicrobial efficacy of GO and PMB-incorporated GO was first characterized by MIC and MBC values against planktonic E. coli and P. aeruginosa. As shown in Table 1, the MIC and MBC of pristine GO and TEG functionalized GO were > 100 µg/mL, hence we did not document any antimicrobial effect of GO alone (Table 1). Bacteriostatic and bactericidal efficiency was established after PMB adsorption. The bacteriostatic effect of GO-PMB was stronger than GO-TEG-PMB against P. aeruginosa. There was no difference in the bactericidal efficiency of GO-PMB and GO-TEG-PMB against both tested bacterial strains (Table 1).

Based on MIC and MBC values, concentrations of test agents were selected for time kill assays gain more detailed insight into the antimicrobial effect. GO and GO-TEG did not fully inhibit the growth of E. coli and P. aeruginosa, but the cell concentration was 10 folder lower than the untreated controls after 24 h incubation (Figure 5). By contrast, the treatment with GO-TEG-PMB significantly reduced the number of viable bacterial cells already after 4 h of incubation. The concentration-dependent bactericidal activity of GO-TEG-PMB was also clearly visible in the time killing assay after 24 h of treatment. Consistent with the MIC and MBC results, P. aeruginosa was more sensitive to the tested agents than E. coli. The viability of P. aeruginosa was drastically decreased by GO-PMB (3 log CFU) and GO-TEG-PMB (5 log CFU) after 24 h of treatment at 2×MIC concentration of tested agents (Figure 5A). In comparable conditions, the viability of E. coli was reduced only by 1 and 2 log of CFUs, respectively, by GO-PMB and GO-TEG-PMB (Figure 5B).

In order to determine the antibiofilm activity, preformed 24 h old biofilms of E. coli and P. aeruginosa were treated with all the tested materials. After the 24 h of treatment with each tested agent, antibiofilm activity was examined by means of CFU counting, live/dead viability staining, and scanning electron microscopy (Figure 7). The viability of both E. coli and P. aeruginosa biofilms were not significantly affected by GO and GO-TEG. By contrast, GO-PMB and GO-TEG-PMB significantly altered the viability of biofilms of both tested stains. Furthermore, the reduction of a number of biofilm cells was observed to be concentration dependent, since 2×MIC reduced the viability of bacteria significantly higher than 1×MIC (Figure 6A). The antibiofilm effect of GO-TEG-PMB was significantly stronger than that of GO-PMB. Interestingly, the increase of the GO-TEG-PMB concentration to 2×MIC was more effective against the E. coli biofilms. This does not follow the trend observed with planktonic bacterial cultures. This difference is likely due to the difference in density and composition of the biofilms matrix of respective bacterial strains, reflecting on their resilience. To confirm the results obtained from CFU counting, control, and treated biofilms were stained with live/dead viability staining and examined using a fluorescence microscope. Figure 6B shows the representative images acquired after the live/dead staining where green color depicts the live cells and red color depicts the dead cells. A higher density of dead cells was observed in biofilms treated with 2×MIC of GO-PMB and GO-TEG-PMB compared to controls. Both P. aeruginosa and E. coli biofilms were more strongly affected by GO-TEG-PMB (green to red ratios should be compared). These observations correlated well with the findings from CFU counting. Furthermore, to examine the effect of the treatment on the architecture of the biofilm matrix and cell morphology, biofilms were examined by SEM (Figure 6C). Both GO-PMB and GO-TEG-PMB were found to significantly alter the biofilm organization and deactivate bacterial cells by disrupting the cell membranes. Both tested materials at a 2×MIC clearly destroyed the biofilms of both P. aeruginosa and E. coli, leaving either a fraction of altered microcolonies or single cells disconnected from the complex community. Most of the visible cells in such microcolonies or individual cells were either morphologically altered or collapsed due to cell membrane damage leading to leakage of intracellular components.

Next, we asked whether the strong biofilm disruptive activity of GO-TEG-PMB can be useful in terms of preventing biofilm formation on typical biomedical devices. To test that, small pieces of catheter tubes were coated with individual test materials. Representative photographs of test agent coated catheters are presented in Figure 7A. The coated catheters were exposed to bacterial cultures (24 h) for biofilm formation and the adhesion of bacterial cells on the catheter surfaces was examined by SEM (Figure 7B). As shown in SEM images, bacterial adhesion and biofilm formation of both P. aeruginosa and E. coli on the surface of pristine GO- and GO-TEG-coated catheters appear to be similar to the control non-coated surface. By contrast, bacterial adhesion was drastically reduced on catheters coated with GO-PMB and GO-TEG-PMB. In addition to preventing bacterial adhesion, GO-PMB and GO-TEG-PMB coatings seemed to also disrupt the adhered bacterial cells.