Electrochemical Regulation of Antibacterial Activity Using Ferrocene-Containing Antibiotics
Song Shen, Yiming Huang, Anran Yuan, Fengting Lv, Libing Liu, Shu Wang
Abstract
Open AccessCCS ChemistryCOMMUNICATION1 Nov 2021Electrochemical Regulation of Antibacterial Activity Using Ferrocene-Containing Antibiotics Song Shen, Yiming Huang, Anran Yuan, Fengting Lv, Libing Liu and Shu Wang Song Shen Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 College of Pharmaceutical Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 , Yiming Huang Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 , Anran Yuan Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 College of Pharmaceutical Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 , Fengting Lv Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 , Libing Liu Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 and Shu Wang *Corresponding author: E-mail Address: [email protected] Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 https://doi.org/10.31635/ccschem.021.202000570 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The widespread use of antibiotics causes the accumulation of a large amount of antibiotics in the environment. Excessively active antibiotics in the environment results in the emergence of bacterial resistance. Building smart antibiotics capable of reversible regulation between active and inactive states on demand is a promising approach to address this issue. Herein, a ferrocene-containing quaternary ammonium compound has been developed for electrochemical redox-controlled bacterial inhibition. The reversible switch of the reduced and oxidized ferrocene groups between hydrophobic and hydrophilic states triggers the assembly and disassembly of the micelles while modulating the interactions of antibiotic molecules with the bacteria membrane, providing a new way to regulate antibacterial activity. In addition, the alternate use of reduced and oxidized antibiotics exhibits a favorable effect in preventing bacterial resistance. Thus, an unconventional strategy is offered to prevent the build-up of active bactericide in the environment and decrease bacterial resistance. Download figure Download PowerPoint Introduction Antibiotics have proven to be effective tools for the treatment of bacterial infections. However, the widespread misuse of antibiotics in healthcare and agriculture causes noticeable resistance due to spontaneous bacterial mutation and horizontal gene transfer.1–3 Although massive efforts have been done to counteract this resistance, such as unnecessary medication reduction, antibiotic combinations, and new antibiotics discoveries,4–7 it is still challenging to completely avoid the emergence and spread of the resistance due to the strong adaptability of the bacteria. To prevent the resistance from evolving, there is an urgent need to develop unconventional strategies to control antibiotic functions and realize on-demand bacterial inhibition.8,9 Building smart antibiotics capable of reversible switching between the active and inactive state in response to external stimuli offers novel concepts to avoid accumulation of the active drug in the environment. Recently, light irradiation has been applied as an external stimulus to regulate the performance of biomedicine and antibacterial activity of biotics.9–11 Upon irradiation, the light-sensitive α-lactam antibiotic can be destroyed to prevent accumulation of the drug in the environment.10 Meanwhile, reversibly photoswitchable antibacterial agents with photoisomeric structures have been designed to realize selective activation/inactivation upon light irradiation.9 In addition, supramolecular assembly and disassembly can be used to regulate the antibacterial activity of biotics.12–15 Once the antibiotic forms a supramolecule, the antibacterial activity is turned off, while antimicrobial activity is recovered with the disassembly of the supramolecule.12 Electric fields are another promising external trigger for the control of antibiotic activity because it can be easily controlled and is relatively noninvasive. However, electric field-responsive antibiotics have seldom been reported yet. In this work, we present an electrically switchable bactericide for active, spatial, and temporal control of the antibacterial activity. The antibacterial agent is based on a cationic "redox-switchable" ferrocenyl quaternary ammonium (QA) compound [(12-ferrocenyl)benzalkonium bromide (FBZK)] that kills the bacteria via QA group penetration into the cell membrane (Scheme 1a).16 At a concentration over the critical micelle concentration (CMC), the reduced FBZK (ReFBZK) self-assembles into micelles, which hinders penetration and leads to a remarkable decrease in antibacterial activity (Scheme 1b).17 The addition of β-cyclodextrin (βCD), which can form a supramolecule with ReFBZK, will further reduce the toxicity. The ferrocenyl group positioned at the end of the alkyl chain is redox-active and can be oxidized to realize the transition from the hydrophobic to hydrophilic state,18,19 allowing for micelle disassembly, and enhanced bactericidal effects. When the concentration is below the CMC, the situation is reversed in that ReFBZK with a hydrophobic tail is more readily able to penetrate into the membrane than oxidized FBZK (OxFBZK) with two hydrophilic heads, exhibiting better antimicrobial activity (Scheme 1c). In this situation, the addition of βCD can inactivate the ReFBZK, providing another strategy to detoxify antibiotics. Scheme 1 | (a) Schematic illustration of the redox reaction of FBZK. (b) The assembly and disassembly of the FBZK micelles caused by the reduction and oxidation at a concentration over the CMC. The assembly of FBZK micelles results in fewer antibiotic molecules to bind to bacteria (e.g., E. coli), while disassembled OxFBZK can significantly enhance the antibiotic concentration on the bacterial membrane. The encapsulation of ReFBZK by βCD remarkably decreased the toxicity. (c) At a concentration below the CMC, ReFBZK with a hydrophobic tail can more easily penetrate the membrane, resulting in stronger toxicity (e.g., S. aureus). Download figure Download PowerPoint Results and Discussion The FBZK was synthesized following the route shown in Supporting Information Scheme S1. The appearance of aqueous FBZK solutions at a concentration of 20 mg/mL is shown in Figure 1a. The ReFBZK solution exhibited a yellowish color with maximum absorption wavelength at 448 nm. The ReFBZK solution was oxidized in phosphate-buffered saline (pH 5.5) by cyclic voltammetry at 25 °C. A platinum plate with a projection area of 0.2 cm2 was used as the working electrode. A platinum wire and Ag/AgCl electrode were used as the counter and reference electrodes, respectively. After oxidization, the yellowish solution of ReFBZK turned to bright green, together with the absorption emergence at 635 nm (Figure 1a). Meanwhile, we also monitored the size change of the ReFBZK and OxFBZK solution. As shown in Figure 1b, the average diameters of the ReFBZK and OxFBZK were 58 and 2.8 nm, respectively. The remarkable decrease in size after oxidation indicated micelle disassembly. The morphology of the FBZK was characterized by transmission electron microscopy (TEM). At a concentration greater than the CMC, the ReFBZK assembled into micelles, demonstrating a spherical structure with relatively uniform sizes in the range of 20–60 nm (Figure 1c). The energy-dispersive X-ray spectroscopy (EDX) mapping results confirm the presence of C, O, Fe, and Br in the ReFBZK micelles (Figure 1d). After electrochemical oxidization, the micelles disappeared and only ultrasmall nanoparticles with an average diameter of ∼1.5 nm were observed, which should be the single OxFBZK nanocrystals (Figure 1e). Figure 1 | (a) UV–vis absorption spectra of FBZK before and after electrochemical oxidation for 1, 2, and 3 h. (b) Particle size distribution of ReFBZK and OxFBZK measured by DLS. TEM image (c) and the corresponding EDX mapping (d) of ReFBZK micelles. (e) TEM image of the OxFBZK. Download figure Download PowerPoint The CMC of the antibiotic was determined spectrophotometrically using Eosin Y dye as the indicator ( Supporting Information Figures S1 and S2).20 As shown in Figure 2a, the CMC value of the ReFBZK in water was 4.5 μg/mL. After oxidization, the CMC increased to 13 μg/mL due to the hydrophobic–hydrophilic switch of the ferrocenyl group. It has been reported that the CMC is related to antibacterial activity.21 When the concentration is under the CMC, the antibiotics are soluble and participate in the antibacterial activity. Greater than the CMC values, the antibiotics assemble into micelles and are no longer active in destroying microbials. Hence, we hypothesize that the antibacterial activity of the FBZK is influenced by CMC and can be controlled by the electrochemical redox reaction. To validate this hypothesis, we investigated the minimal inhibitory concentration (MIC) of FBZK toward Escherichia coli and Staphylococcus aureus (Figure 2b). The MIC of ReFBZK for E. coli was 25 μg/mL, which was much higher than the CMC (4.5 μg/mL). In contrast, the OxFBZK demonstrated a relatively lower MIC of 12.5 μg/mL for E. coli, which was below the CMC (13 μg/mL). The enhanced antibacterial activity should be attributed to the hydrophobic–hydrophilic transition and micellar disassembly. Meanwhile, the increased charge density of OxFBZK should enhance the binding effect to negatively charged bacteria and further improve the antibacterial activity. For S. aureus, the situation was reversed in that ReFBZK with a MIC of 2.5 μg/mL was more powerful than the oxidized counterpart (with a MIC of 5 μg/mL). The possible reason is that the ReFBZK with one hydrophilic head and hydrophobic chain can more easily penetrate the cell membrane than the antibiotic with two hydrophilic heads. Figure 2 | (a) The CMC of the ReFBZK and OxFBZK in the absence and presence of βCD with different concentrations (50 and 500 μg/mL). (b) MIC of ReFBZK and OxFBZK for E. coli and S. aureus after co-incubation for 30 min. (c) Growth curves of E. coli treated with ReFBZK and OxFBZK at a concentration of 12.5 μg/mL, in the presence and absence of βCD (50 μg/mL). (d) Growth curves of S. aureus treated with ReFBZK and OxFBZK at a concentration of 2.5 μg/mL, in the presence and absence of βCD (50 μg/mL). (e) Photographs showing the colony forming units (CFU) of E. coli and S. aureus treated with ReFBZK and OxFBZK with and without βCD (50 μg/mL). Quantitative evaluation of the antibacterial activity of ReFBZK and OxFBZK against E. coli (f) and S. aureus (g) with and without βCD. Error bars represent the standard deviation (SD) of the mean (n = 3). Download figure Download PowerPoint The growth of E. coli and S. aureus could be successfully controlled by the electrochemical redox at concentrations of 12.5 and 2.5 μg/mL, respectively (Figures 2c and 2d). βCD, which could form supramolecular assembly with FBZK, was used to further regulate the antibacterial activity of FBZK. In the presence of βCD, the inhibition effect of FBZK toward E. coli was noticeably weakened due to the supramolecular host–guest interaction between βCD and ferrocene (Figure 2c).22 In contrast, only negligible influence was detected in the OxFBZK group. Similar phenomenon was also observed in the S. aureus group (Figure 2d). However, when the concentration of βCD subsequently increased to 500 μg/mL, the antibacterial activity of ReFBZK and OxFBZK was remarkably inhibited ( Supporting Information Figure S3). The inhibited antibacterial activity should be attributed to the encapsulation of βCD, which hampered the cell membrane penetrability of FBZK. The reversible interaction between βCD and FBZK was investigated by the characterizations of CMC and 1H NMR. The CMC of ReFBZK was obviously increased as βCD concentration increased, while the CMC of OxFBZK was not influenced (Figure 2a). The 1H NMR ( Supporting Information Figure S4) results showed the proton peaks of βCD at 3.92–3.82 ppm and a 0.07 ppm shift toward a lower field after mixing with ReFBZK. Furthermore, the characteristic protons signals of ReFBZK at 3.95–4.06 ppm moved to 4.08–4.21 ppm. These observations imply the formation of the βCD/FBZK complex. Then, we examined the antibacterial activity of the ReFBZK and OxFBZK in the presence and absence of βCD by colony counting assay method. The survival rate of E. coli treated with ReFBZK alone was 22.3%. By contrast, only a survival rate of 0.8% was observed for OxFBZK at the same conditions (Figures 2e and 2f), suggesting the well-controlled antibacterial activity by electrochemical redox. The presence of βCD could remarkably decrease the killing efficiency of ReFBZK from 77.7% to 62.5%, while only a slight influence was shown on OxFBZK. In the S. aureus group, ReFBZK exhibited a much better inhibition effect than OxFBZK due to the hydrophobic chain, which facilitated permeation (Figures 2e and 2g). Due to the formation of the supramolecular complex, the killing effect of ReFBZK against S. aureus was weakened more remarkably by βCD than that of OxFBZK. The switchable ability in killing bacteria was illustrated by alternating oxidization and reduction of FBZK followed by the incubation with bacteria ( Supporting Information Figure S5). The antimicrobial function could be reversibly and repeatedly turned on and off by the redox reaction using alternating electric field as an external trigger. QA compounds are generally believed to electrostatically interact with negatively charged bacterial cellular membrane and subsequently penetrate the side chains into the membrane to induce the leakage of the bacterial content. To understand the mechanism of the reversible antibacterial activity, the morphological changes of the bacteria after incubation with ReFBZK and OxFBZK were characterized by scanning electron microscopy (SEM). As shown in Figure 3a, the untreated E. coli and S. aureus were rod-shaped (for E. coli) and spherically shaped (for S. aureus) with an intact and smooth membrane. After treatment with OxFBZK and OxFBZK + βCD, almost all of the E. coli were seriously damaged, as characterized by the distorted bacterial membranes and collapsed bacteria. In contrast, a relatively low toxicity was observed in the ReFBZK and ReFBZK + βCD groups that only a portion of the bacteria was destroyed. In the βCD groups, the bacterial morphology was not influenced, indicating the toxicity was caused by the cationic antibiotic ( Supporting Information Figure S6). As for the S. aureus, ReFBZK displayed greater interference effects to the integrity of the membrane than the OxFBZK. The presence of FBZK on the membrane of the bacteria was also verified by the EDX results ( Supporting Information Figure S7). The antibacterial effect was also evidenced by the live/dead staining assay that the red staining of dead bacteria indicated the damaged membrane, while the live bacteria were stained green ( Supporting Information Figures S8 and S9). Figure 3 | (a) SEM images of E. coli and S. aureus treated with ReFBZK and OxFBZK in the presence and absence of βCD. (b) ζ potentials of E. coli and S. aureus upon the addition of ReFBZK and OxFBZK with and without βCD. (c) Growth curves of ReFBZK resistant E. coli treated with ReFBZK and OxFBZK at concentrations of 30, 60, and 90 μg/mL. (d) Growth curves of ReFBZK resistant S. aureus treated with ReFBZK and OxFBZK at concentrations of 2.5, 5, and 7.5 μg/mL. The data points were represented as the mean ± SD (n = 3). Download figure Download PowerPoint The zeta potential (ζ) of the bacteria was determined to investigate the interactions between the FBZK and two types of bacteria with respect to oxidation and reduction. As presented in Figure 3b, a pronounced increase of the potentials of Gram-negative bacteria E. coli was observed after the treatment of FBZK, while the potentials changes of Gram-positive bacteria S. aureus was not so obvious. This is because the FBZK could insert into the thick, porous wall and be hidden in the wall of S. aureus, creating insignificant changes in potential.23 For the case of E.coli, FBZK was restrained to the relatively thin outer wall, resulting in a remarkably positive potential shift. Although the βCD could decrease the antibacterial activity of FBZK, no obvious change in potential was observed. These results imply that FBZK binds to the surface of the bacteria in the form of a βCD/FBZK complex instead of penetration. There was no conspicuous difference in the potential of the bacteria between the ReFBZK and OxFBZK treated groups. Then, we investigated the antimicrobial activity of ReFBZK and OxFBZK against the resistant bacteria. The resistant bacteria were obtained by incubating E. coli and S. aureus with ReFBZK at concentrations of 20 and 2 μg/mL for 1 week, respectively. The resistant E. coli was tolerant to ReFBZK and could not be completely inhibited even at a concentration of 90 μg/mL (Figure 3c). In comparison, OxFBZK was highly efficient and could kill the bacteria at a concentration of 30 μg/mL. As for S. aureus, OxFBZK also exhibited greater antibacterial activity than ReFBZK (Figure 3d). The above results suggest that smart antibiotics not only realize on-demanding bacterial inhibition but are also capable of preventing resistance emergencies. Conclusion We have demonstrated a proof of concept using a redox-active cationic antibiotic to regulate antibacterial activity with an alternating electric field. The ferrocene-containing antibiotic could be controlled to be active and inactive by redox-induced transition between hydrophobic and hydrophilic states. In the presence of βCD, the antibacterial activity could be further modulated by the formation of supramolecular assembly. Meanwhile, the OxFBZK indicated a reversible effect for the bacteria resistant to ReFBZK. This work presents an effective strategy in controlling bacterial growth and the potential for the prevention and treatment of bacterial resistance. 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