Synergistic Antibacterial Activity of Black Phosphorus Nanosheets Modified with Titanium Aminobenzenesulfanato Complexes

Introduction: A two-dimensional (2D) antimicrobial nano-agent is synthesized by incorporating the titanium aminobenzenesulfanato complexes (Ti-SA₄) onto black phosphorus nanosheets (BPs). The strong P-Ti coordination between Ti-SA₄ and BPs results in the high loading capacity (~43%) of Ti-SA₄ onto BPs and enhances the stability of BPs against oxidation. Compared to bare BPs and Ti-SA₄, the Ti-SA₄@BPs exhibit improved antibacterial efficacy and most of the bacteria are inactivated within 3 h with a dose of only 50 μg/mL. The underlying antibacterial mechanism is investigated. This proposed Ti-SA₄@BPs have great potential in clinical applications and the results provide insights into the design and synthesis of 2D antibacterial nano-agents. 
Materials and Methods: The BP crystals were dispersed in the NMP solution (25 mL) and treated in an ultrasonic ice bath at 40 kHz frequency. The solution was centrifuged at 4,000 rpm for 10 min after exfoliation to remove the unexfoliated bulk and the supernatant containing the BPs was stored. 
To synthesize Ti-SA₄, a solution of 4-amino-3-methylbenzenesulfonic acid in EtOH were added with Ti(OiPr)₄. The molar ratios of 4-amino-3-methylbenzenesulfonic acid to Ti(OiPr)₄ was 4/1. The mixture was heated at 50 °C for 5 h under argon. After removing the solvent, the crude mixture was passed through a bed of silica gel and TiSA₄ was obtained as a yellow solid. 
To fabricate Ti-SA₄@BPs, the BPs were dispersed in NMP with the addition of an excessive amount of Ti-SA₄. The mixture was stirred in darkness under the protection of Ar environment for 20 h. The mixture was centrifuged at 12,000 rpm for 20 min and the precipitated Ti-SA4@BPs were collected. 
Results and Discussion: Ti-SA₄ is synthesized by a reaction between 4-amino-3-methylbenzenesulfonic acid (SA) and titanium tetraisopropoxide [Ti(OiPr)₄].Both the ¹H and ¹³C NMR spectra confirm successful synthesis of Ti-SA₄. After liquid exfoliation of BPs, the Ti-SA4@BPs are prepared by loading Ti-SA₄ onto BPs via surface coordination. The samples are characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). As shown in Figures 2a and 2b, the Ti-SA₄@BPs exhibit the typical 2D morphology with an average lateral size and thickness of ~220 nm and ~5 nm, respectively. The high-resolution TEM (HR-TEM) image of Ti-SA₄@BPs (inset in Figure 2a) reveals lattice fringes of 2.5 Å corresponding to the (014) plane of BP crystal. 
Two common bacteria, E. coli (Gram-negative bacterial strain) and S. aureus (Gram-postive bacterial strain), are employed to evaluate the antibacterial activity of Ti-SA₄@BPs. For comparison, the antibacterial performance of the bare BPs and Ti-SA₄ with the same corresponding amount of Ti-SA₄@BPs are also studied. 5×10⁶ CFU/mL of each bacterial strain are incubated with 15 μg/mL Ti-SA₄, 35 μg/mL BPs, or 50 μg/mL Ti-SA₄@BPs respectively, and subjected to biological assays. Besides Live/Dead staining, the antibacterial efficacy is quantitatively determined and the results are displayed in Figure 3. The intense green fluorescence from E. coli and S. aureus in the blank group indicate that almost all the bacteria are alive. After Ti-SA₄ or BPs treatment, green fluorescence recedes, whereas red fluorescence increases implying that the bacteria are partly sterilized. In comparison, the TiSA₄@BPs group delivers the most efficient antibacterial performance, from which intense red fluorescence and aggregated dead bacteria are readily observed. 
In view of the outstanding antibacterial properties of Ti-SA₄@BPs, the morphological changes of bacteria after different treatments are examined by scanning electron microscopy (SEM). As illustrated in Figure 3, the untreated E. coli and S. aureus show the typical bacterial morphology with a smooth surface. With regard to the Ti-SA₄ and BPs groups, most of the bacteria retain the normal shape indicating that the single Ti-SA₄ or BPs treatment is not effective enough to produce bacterial membrane damage. In contrast, the bacteria treated by Ti-SA₄@BPs are wrinkled and damaged membranes are observed. The Ti-SA₄ ligands designed for this study derive from antimicrobial sulfonamides, which are used in clinical treatment of infectious diseases. Nevertheless, similar to most sulfa drugs, Ti-SA₄ suffers from low antibacterial efficacy. By combining with BPs coordination, the antibacterial performance of Ti-SA₄@BPs is improved significantly compared to Ti-SA₄ for the same concentration. The excellent performance stems from strong P-Ti coordination which leads to local enrichment of Ti-SA₄ on the surface of nano-sized BPs and subsequently facilitates the interaction between Ti-SA₄ and microorganisms to deliver better therapeutic effects. From another point of view, Ti-SA₄@BPs with a positive surface potential are much more accessible than bare BPs with negative potential to bacteria with negatively-charged membranes. The high affinity between Ti-SA₄@BPs and bacteria isolate the bacteria from the survival condition and the sharp edges of 2D Ti-SA₄@BPs can penetrate the phospholipid menbranes of bacteria to destroy the membranes causing eventual bacteria death. 
Conclusions: In conclusion, a 2D antimicrobial nano-agent is designed and fabricated by loading the sulfonamide metal complex onto BPs. Strong P-Ti coordination gives rise to efficient loading (~43%) of Ti-SA₄ onto BPs and enhances the stability of BPs against oxidation. The Ti-SA₄@BPs exhibit markedly improved antibacterial efficacy compared to bare Ti-SA₄ and BPs. Using a dose of about 50 μg/mL, the Ti-SA₄@BPs are capable of inactivating most of the bacteria within 3 h. Further investigation of the antibacterial mechanism demonstrates that not only the coordination strategy increases the therapeutic effect of Ti-SA₄ by locally enriching the antibacterial agents on the surface of nano-sized BPs, but also the Ti-SA₄@BPs with surface positive charges facilitate the interaction with negatively-charged bacteria result in the destruction of bacterial membrane, reduced protein synthesis, and irreparable DNA damage. Considering the biodegradability and biocompatibility of BPs, the 2D Ti-SA₄@BPs are promising in clinical applications. Furthermore, the results provide insights into the design and synthesis of new 2D nano-agents that can meet the multifarious and rigorous requirements of biomedicine.