Self‐reporting fluorescence deciphers the antibacterial nature of cationic amphiphiles: Monomer or aggregate?

Cationic amphiphile aggregates exhibit superior antibacterial activity than monomers. However, the antimicrobial mechanism of aggregates has not been well understood because it is difficult to distinguish and monitor aggregate and monomer in antimicrobial process. Herein, three bola‐type cationic amphiphiles with aggregation‐induced emission property have been developed to show distinguishable fluorescence in their monomer and aggregate. The hydrophilicity of monomer and the stability of aggregate are finely tuned by tailoring the linkers between two quaternary ammonium end groups and tetraphenylethylene skeleton. The sensitive fluorescence switching of monomer and aggregate achieves the quantitative monitoring of dynamic interaction of three amphiphiles with bacteria. The aggregates with cationic charges first attach to bacterial surface, and the monomers subsequently dissociate from aggregates to penetrate bacterial membrane. Further, our results reveal the vital role of stability of aggregates during antimicrobial process, shedding light on the rational design of high‐efficient antimicrobials.

process on the bacterial surface. [8,15,16]Moreover, numerous aggregate structures with different sizes, shapes, and stability can be created by tailoring the moiety and composition of amphiphilic molecules, providing a wide window for the construction of highly effective antimicrobials. [8,17,18]n the other hand, the understanding of antimicrobial mechanism of cationic amphiphile aggregates has lagged far behind their antimicrobial applications.Some studies found that the antimicrobial activity of aggregates showed size-/shape-dependence. [12,17,19,20] However, contradictory results occurred frequently, where aggregates with the same size and shape exhibited significantly different antimicrobial activity. [13]A possible explanation is that the dynamic nature of aggregates may lead to their disassembly into monomers upon contact with bacteria, affecting the antimicrobial process. [21]Therefore, real-time quantitative monitoring of the dynamic interaction of aggregates and monomers with bacteria is essential to understand the antibacterial nature of cationic amphiphiles.
Self-assembled cationic amphiphiles have been found to bind with the bacterial surface and cause the membrane disruption using electron microscopy, [22] isothermal titration calorimetry, [9,13] fluorescent technology [11] and so on.28] However, conventional fluorophores have rigid and planar structures that cause them to experience a loss of fluorescence when aggregated, known as aggregation-caused quenching (ACQ). [26,29]The lack of fluorescence of amphiphile aggregates makes it difficult to obtain fluorescence information on the interaction between bacteria with amphiphile aggregates.Therefore, it remains a challenge to selectively identify and detect amphiphile aggregates and monomers in the antimicrobial process.
[32][33][34] Different from the conventional ACQ fluorophores, the AIEgens show usually weak emission as the isolated molecules with free intramolecular motions, but become highly emissive upon aggregation based on the restriction of intramolecular motion mechanism. [35]Depending on the restricted level of their intramolecular motions by the local surroundings, the AIEgens show the different fluorescence intensities.Concomitantly, their twisted molecular conformation may also be adjusted, which would cause the change in maximum emission. [36,37]These features make AIEgens an excellent indicator for in situ identification of the aggregated and monomeric states of amphiphiles.In combination with the different microenvironments of amphiphile monomers, amphiphile aggregates, and bacteria, [23,38] it is reasonable to obtain clearly distinguishable fluorescence signals by rationally incorporating the AIE attribute into the cationic amphiphiles.
To achieve self-reporting fluorescence of amphiphile monomers and aggregates, three AIE-active cationic bolatype amphiphiles were developed, where tetraphenylethylene (TPE) moiety was introduced as a hydrophobic fluorescence skeleton and attached to two hydrophilic ammonium heads via three tailored linkers (i.e., oxyethyl, di(oxyethyl), and oxyhexyl).The obtained amphiphiles with gradually increasing hydrophobicity were named as TO2C, T2OE, and TO6C, respectively (Figure 1).The three AIE-active amphiphiles can clearly self-report their two states (aggregates and monomers) by the variation of their fluorescence intensity and wavelength.Taking TO2C as an example, its blue fluorescent aggregates were observed in real time under fluorescence microscopy to rapidly attach to the bacterial surface and then disassemble into the cyan fluorescent monomers for inserting into the bacteria (Figure 1).Interestingly, visualization of the antimicrobial dynamics demonstrated that the three amphiphile aggregates were unable to penetrate and disrupt bacterial membranes by themselves.The aggregates disassembled into monomers upon contact with bacteria, and their disassembly rate determined their antimicrobial efficiency, which was associated with their thermodynamic stability.These results experimentally deciphered that the antimicrobial process of the amphiphiles relied on both the disassembly of aggregates and the membrane penetration of monomers.

The photophysical and self-assembly properties
To systemically tune the hydrophilicity of cationic amphiphiles and the stabilities of formed aggregates, T2OE, which employs di(oxyethyl) as a linker between TPE skeleton and ammonium heads, was designed and synthesized by facile synthetic routes (Figure S1).The chemical structure of T2OE was fully characterized by 1 H NMR, 13 C NMR and HRMS spectra (Figures S2-S4).TO2C and TO6C were synthesized according to the previous report, [39] and their chemical structure were characterized by 1 H NMR and 13 C NMR (Figures S5-S8).
One main absorption peak at about 318 nm was observed for TO2C, T2OE, and TO6C in DMSO solution (Figure S9).With the incorporation of TPE moiety, a typical AIE characteristic was observed for three amphiphiles.Taking TO2C as an example (Figure S10a,b), TO2C emits weakly in DMSO/toluene mixtures with toluene fractions below 85 vol%.Upon increasing the toluene fraction above 88 vol%, a strong emission peak at about 467 nm appears and the emission intensity increases with the increase of toluene content, showing an obvious AIE phenomenon due to the formation of aggregates with submicrometer size (Figure S11).
Based on the obvious change of emission intensity upon aggregation, the critical aggregation concentrations (CACs) of three amphiphiles can be determined.The corresponding fluorescence spectra of three amphiphiles with different concentration in phosphate buffered saline (PBS) solution were recorded (Figure S12).It was observed that their emission intensity increases with increasing the concentration.From the plots of maximum emission intensity against the concentrations (Figure 2A-C), the CAC values of TO2C, T2OE, and TO6C in PBS solution were estimated to be 34.2,3.8, 1.9 μM, respectively.This indicates that the self-assembly ability for three amphiphiles follows a trend of TO2C < T2OE < TO6C, depending on their hydrophilicity with respective calculated n-octanol/water partition coefficient (ClogP) values of 0.75, 1.12, and 2.77.Compared with TO2C and TO6C, T2OE still shows weaker emission after aggregation (Figure 2B and Figure S12b), suggesting the formation of loose structure.Besides the discrepancy in emission intensity, three amphiphiles show also obvious variation of emission wavelength after aggregation.As shown in Figure 2A, a significant blue-shift of the emission peak from 480 nm to approximately 457 nm was observed for TO2C at the concentration above its CAC.Similarly, T2OE also shows a blue-shift emission peak but smaller than that of TO2C, changing from 473 to 470 nm upon aggregation (Figure 2B).As for TO6C bearing a longer hydrophobic linker, the occurrence of aggregation causes a red-shift of its emission wavelength from 477 to 481 nm (Figure 2C).This change in emission wavelength of three amphiphiles before and after aggregation, especially TO2C, facilitates us to readily distinguish between their aggregated and monomeric states when observing their interaction with bacteria.
Above CACs, TO2C, T2OE, and TO6C all form the rectangular aggregates but differed by molecular packing of TPE core within aggregates.Transmission electron microscopy images of Figure 2D and dynamic light scattering results of Figure 2G show that TO2C forms the rectangular aggregates with an average diameter of around 200 nm and a narrow size distribution (polydispersity index, PDI = 0.13) in PBS solution.Also, TO6C forms about 200 nm of rectangular aggregates (Figure 2F) with a relatively narrow size distribution (PDI = 0.31, Figure 2G).In contrast, the size distribution of the rectangular aggregates formed by T2OE becomes wider with the diameter range of 50-180 nm (PDI = 0.74, Figure 2E,G), consistent with loose aggregate structure reflected by the emission intensity.Despite the different size distribution, the formed rectangular aggregates by three amphiphiles are all crystalline with the regular diffraction points, as confirmed by their transmission electron diffraction patterns (insert, Figure 2D-F), suggesting that three amphiphiles take an ordered molecular packing inside their aggregates.The respective zeta potential values for the aggregates were measured as about 13.8, 7.6, and 18.3 mV for TO2C, T2OE, and TO6C (Figure 2H), which indicates their positively charged surface.
The crystalline aggregates of three amphiphiles show an obvious discrepancy in their emission intensity and wavelength, suggesting the diverse arrangement tightness and configuration of TPE core within their aggregates. [40]As shown in Figure 2I, an emission peak centered at 457 nm was observed for TO2C aggregates, whereas a strong and a large red-shifted emission peak at about 484 nm for TO6C aggregates, and a weaker emission centered at about 470 nm for T2OE aggregates.The difference in emission intensity of aggregates formed under the same concentration indicates the arrangement tightness of three amphiphiles inside decreases in the order of TO6C > TO2C > T2OE.With inserted two long hexyl linkages, TO6C molecules are driven to arrange tightly to form the crystalline aggregates by strong hydrophobic interaction, which effectively restricts the intramolecular rotation of TPE core and thus gives the strong emission. [41]ccompanied with that, the TPE core of TO6C molecule would be induced to take a more coplanar conformation, accounting for the red-shifted emission in comparison with the other two molecules, as supported by a red-shifted absorption peak of TO6C aggregates (316 nm) compared with that of TO2C and T2OE aggregates (309 nm, Figure 2I).In addition, the aggregation of TO2C and T2OE also causes an obvious blue shift of their main absorption peak to 309 nm from about 318 nm of their monomers in DMSO solution (Figure S9).This further verifies that the molecular conformations of TO2C and T2OE become more twisted in their aggregated states, which causes the blue-shift in their PL spectra compared with their monomers (Figure 2A,B).Therefore, three amphiphiles adjust themselves to take a different twisted conformation to fit into their surrounding environment, giving rise to diverse emission wavelength in their respective monomeric and aggregated states. [36,40,42]

Antibacterial action of aggregates
The antibacterial activity of TO2C, T2OE, and TO6C was estimated by choosing Gram-positive Staphylococcus aureus (S. aureus), one of main pathogens in hospital and community infections [43][44][45][46] , as the representative.A dose-dependent activity of three amphiphiles against S. aureus was tested by the plate colony-counting method.As shown in Figure 3A and Figure S13, at the concentration below their CAC values, these amphiphiles are not potent enough to kill S. aureus.
Once the concentration increases above their CACs, their killing efficiency toward S. aureus is rapidly boosted.As shown, the killing efficiency of TO2C reaches rapidly from 34.6% to 98.7% as its concentration increases from 30 μM (below its CAC of 34.2 μM) to 50 μM (beyond its CAC), and causes all bacterial death with 100% killing activity at 100 μM (Figure 3A).As for T2OE, at the concentration of 5 μM just beyond its CAC (3.8 μM), the antibacterial activity against S. aureus is rapidly enhanced to 57.7%, and then increased to 79.4% at 20 μM, to 95.3% at 50 μM and to 100% at 100 μM.Similarly, just beyond the CAC of 1.9 μM, the killing efficiency of TO6C is improved to 76.5% at 3 μM, and then increased to 94.0% at 5 μM and to 100% at 10 μM.Furthermore, the minimal bactericidal concentration (MBC), an important parameter to describe the killing efficiency of antimicrobials, is determined for three amphiphiles. [47]As shown in Figure S14, the MBC 50 values (the concentration killing 50% of the bacteria) of TO2C, T2OE, and TO6C are assessed to about 34.5, 11.8, and 2.5 μM against S. aureus, respectively.And their MBC 90 values (the concentration killing 90% of the bacteria) are about 53.6, 35.4 and 3.9 μM for TO2C, T2OE, and TO6C, respectively, which are much lower than that of traditional macrolide erythromycin (Figure S15a,d) and β-lactams cephalothin (Figure S15b,e) with MBC 90 > 100 μM.Also, TO6C shows much higher killing activity against S. aureus than that of aminoglycosides kanamycin with MBC 50 of 14.4 μM and MBC 90 of 34.6 μM (Figure S15c,f).This fully confirms that the cationic amphiphiles are a promising antibacterial alternative.In addition, it was found that both MBC 50 and MBC 90 values of TO2C, T2OE and TO6C are larger than their respective CACs in PBS (34.2, 3.8, and 1.9 μM).These results fully suggest that the formation of aggregates is an essential factor for their antibacterial potency.As reported, the formation of aggregates enhances surface cationic charges and local concentration of amphiphiles, which promotes their electrostatic binding and accumulation on the negatively charged surface of bacteria. [15]Thus, with the strongest self-assembly ability (i.e., the lowest CAC), TO6C exerts the highest antibacterial activity toward S. aureus with a low MBC 90 of 3.9 μM.However, it was noted that, with a CAC value comparable to TO6C, T2OE (MBC 90 = 35.4μM) shows the antibacterial activity toward S. aureus about one order of magnitude lower than that of TO6C (MBC 90 = 3.9 μM).Also, despite the CAC value of TO2C an order of magnitude higher than that of T2OE, the similar killing efficiency was presented, as indicated by their MBC 90 value of 35.4 μM for T2OE and 53.6 μM for TO2C.This means, besides the self-assembly ability, there exist the other essential factors that dictate the antibacterial potency of cationic amphiphiles.
To reveal this, the fluorescence imaging was firstly performed to visualize the interaction of TO2C, T2OE, and TO6C with S. aureus.As shown in Figure 3B, S. aureus was lighted up with a high labeling efficiency by the aggregates of three amphiphiles after incubated for 30 min, respectively.Moreover, it was observed that TO2C, T2OE, and TO6C penetrate into the S. aureus, which was further verified by the zeta potential results (Figure 3C).The addition of positively charged TO2C (13.8 mV) and T2OE aggregates (7.6 mV) does not lead to the obvious change in the surface zeta potential of S. aureus.A slight positive potential shift of S. aureus from −11.13 to −8.6 mV is caused in presence of TO6C aggregates with large positive-charged surface (18.3 mV), possibly attributed to the ammonium heads of TO6C molecules partially exposed on the surface of S. aureus due to their bearing two long hexyl linkages.No obvious surface potential change of S. aureus caused by three cationic amphiphiles suggests that they insert into the bacterial membrane or entered the bacteria. [48]Given that it is impossible for the aggregates with submicrometer size to directly enter the bacteria with the size of around 1 μm, there must exist the process where the aggregates dissociate into the monomers after electrostatically attaching to the negatively charged bacterial surface and then the monomers penetrate into the bacteria.To confirm this, the fluorescence spectra of TO2C, T2OE and TO6C aggregates before and after interaction with S. aureus for 30 min were collected (Figure 3D and Figure S16), since that our system allows the identification of both aggregated and monomeric states based on the variation of emission wavelength.As shown, after the addition of S. aureus, a large red-shift from 457 to 470 nm along with an enhanced fluorescence intensity was observed for TO2C (Figure 3D).A smaller red-shift from 470 to 475 nm along with a remarkable increase of emission intensity is caused for T2OE (Figure S16a) and a blue-shift from 483 and 480 nm but with a slight enhancement in emission intensity was observed for TO6C (Figure S16b).These emission shifts are almost consistent with that obtained by their in situ spectra on the cell level from the confocal laser scanning microscopy (CLSM) (Figure S17).Referring to the corresponding fluorescent information of three amphiphiles at their aggregated and monomeric states as discussed above, the imaging and spectra results support the hypothesis that the aggregate-monomer equilibrium of three amphiphiles is pushed toward the monomeric state upon contact with bacteria, and then the monomers penetrate into bacteria.
Furthermore, the co-staining experiments of S. aureus incubated by the amphiphiles for 30 min and then stained by propidium iodide (PI) show that the red signal from PI was observed for S. aureus with the sky-blue emission (Figure S18).Since PI selectively enters the cell protoplasm of dead microbes with destroyed cell membrane along with red emission, [49] the co-staining results reveal that the monomers dissociated from the aggregates of three amphiphiles disrupt the bacterial membrane to cause the death of S. aureus, which was further confirmed by the scanning electron microscopy (SEM) images (Figure 3E).For the control group without any treatments, the intact and smooth S. aureus were observed.In contrast, after treated with TO2C, T2OE, and TO6C, the structures of S. aureus are collapsed and merged, and the bacterial membranes are obviously disintegrated.Thus, TO2C, T2OE, and TO6C exert the antibacterial activity presumably through that their aggregates work as a monomer reservoir and disassemble into the monomers to intercalate into and destroy the bacterial membrane. [9]This gives us a hint that the dissociation tendency of aggregates into monomers (i.e., the stability of aggregates) as well as the ability and dynamics of monomers inserting into and disrupting bacterial membrane are also determinant factors for antibacterial potency of cationic amphiphiles, [50] besides their self-assembly ability, as discussed in the following.

Dynamics of aggregate-bacteria interaction
To shed more light on the antimicrobial action of cationic amphiphiles, the interaction of aggregates with bacteria as a function of time was explored using TO2C with obvious emission wavelength variation upon disassembly as a representative (the concentration of 100 μM with the killing activity of 100% against S. aureus after incubation for 30 min was chosen).First, the fluorescence spectra of TO2C were recorded in the presence of S. aureus over time.As shown in Figure 4A, the addition of S. aureus causes the gradual enhancement in fluorescence emission of TO2C with the extension of time.Moreover, an obvious emission red-shift of TO2C was observed after adding S. aureus, which corresponds to some disassembly of aggregates into monomers.Compared with that of TO2C aggregates centered at 457 nm, the emission peak of TO2C immediately red-shifts to 463 nm in the presence of S. aureus (0 min, Figure 4B), suggesting the rapid disassembly of TO2C aggregates into the monomeric states.After incubation for 5 min, the emission red-shifts to about 468 nm and then almost remains unchanged (Figure 4B).This means that the disassembly process of TO2C aggregates upon contact with S. aureus can reach the equilibrium within 5 min.
To further monitor the time course of interaction of TO2C aggregates with bacteria, the fluorescence imaging of S. aureus after incubated with TO2C aggregates for the different time (Figure S19) and co-stained with PI was performed (Figure 4C).It was observed that S. aureus was immediately lighted up after adding TO2C aggregates with a high labeling rate of nearly 100% (Figure 4C,D).Meanwhile, the staining rate of S. aureus by PI reaches 85.4% (the TO2C and PI mission do not interfere with each other under the used imaging conditions, as shown in Figure S20), indicating that nearly 90% of S. aureus was killed at 0 min.This was further verified by the plate colony-counting method, where 100 μM of TO2C aggregates shows 94.0% of killing activity at 0 min (Figure 4D and Figure S21).Meanwhile, the surface zeta potential of S. aureus was measured to be about −8.3 mV in the presence of TO2C aggregates at 0 min (Figure 4E), close to that of S. aureus itself (−11.0 mV).This suggests that TO2C shows a fast dynamics of entry into bacteria, which contributes to its fast dynamics of killing S. aureus.Upon extending the incubation time, the stained S. aureus by TO2C become gradually brighter (Figure 4C and Figure S22), which is almost consistent with that of their bulk solutions (Figure 4A).This suggests that more TO2C aggregates disassemble into monomers to enter the bacteria, as verified by the further red-shifts of TO2C emission over time (Figure 4B) and further decrease in zeta potential of S. aureus close to that of S. aureus itself over time (Figure 4E).Meanwhile, the staining efficiency of PI for S. aureus increases to 92.8% at 5 min and 94.0% at 10 min.The corresponding tested killing activity reaches 99.2% at 5 min and 100% at 10 min (Figure 4D).This indicates that the interaction of TO2C aggregates with S. aureus shows a fast dynamics and equilibrates at about 5-10 min.

Dictation of aggregates stability on antimicrobial dynamics
Given the packing tightness of three amphiphiles within aggregates in the order of TO6C > TO2C > T2OE discussed above, the thermodynamic stability of their aggregates should also follow this rule, as revealed by their surface potentials.Colloidal systems with the larger absolute zeta potential values are generally demonstrated to be more stable. [51]ccordingly, TO6C aggregates with a high ζ-potential of 18.3 mV show the highest stability, followed by TO2C (13.8 mV) and T2OE (7.6 mV).This means, the disassembly tendency of aggregates of three amphiphiles follows the order of T2OE > TO2C > TO6C and thus present diverse dynamic responsiveness when interacting with bacteria.
With the less stable aggegates than that of TO2C, T2OE shows also a fast dynamics of penetrating into bacteria.As shown in Figure 5A,C, S. aureus was immediately lighted up after adding T2OE aggregates with a high labeling rate of nearly 100%.This is ascribed that less stable T2OE aggregates readily disassemble into monomers at the bacterial membrane of S. aureus followed by entry into the S. aureus, as indicated by the immediate emission red-shift from 470 to 479 nm in the presence of S. aureus (0 min, Figure 5B and Figure S23).But at this time, the staining rate of S. aureus by PI is 49.6% (Figure 5A,C, T2OE mission has no effect on the PI fluorescence signal (Figure S24)), much lower than that in the case of TO2C under the same condition (85.4%).Correspondingly, the lower killing activity against S. aureus was observed for T2OE aggregates (63.7%, Figure 5C and Figure S25) than TO2C aggregates (94.0%) at 0 min.This may be ascribed to the ability of T2OE to destroy bacterial membrane is weaker than that of TO2C because the alkoxy chain is essentially weaker than the alkyl chain in destroying membranes. [8,52]The stained S. aureus by T2OE become gradually brighter over time (Figure 5A, Figures S23, S26, and S27), suggesting that more T2OE monomers penetrate into the S. aureus, as verified by the emission shifts to about 475 nm after incubation for 5 min and then almost keeps constant (Figure 5B) and further decrease in zeta potential of S. aureus close to that of itself (Figure 5D).Concomitantly, the staining efficiency of PI for S. aureus increases to 90.7% and the killing activity of T2OE against S. aureus reaches 95.8% at 5 min (Figure 5C and Figure S25), lower than that of TO2C (99.2%).At 10 min, the killing efficiency of T2OE against S. aureus reaches 100% but the staining efficiency of PI is still at about 91%, lower than in the case of TO2C (94.0%).These results confirm that the interaction of T2OE aggregates with S. aureus also shows a fast dynamics and equilibrates at about 5-10 min but the ability of T2OE monomers in disrupting bacterial membrane is weaker than that of TO2C.Consequently, with the stronger self-assembly ability and less self-assembly stability, T2OE shows a similar killing efficiency against S. aureus to that of TO2C.
In contrast, TO6C, which forms the most stable aggregate, exhibits no obvious entry into S. aureus at 0 min (Figure 5E), along with the negligible staining efficiency and a low killing activity of 8.8% against S. aureus (Figure 5G and Figure S28).Thus, unlike the case of TO2C and T2OE, TO6C shows a slower insertion dynamics because the strong stability of their aggregates reverses the dissociation tendency of aggregates to monomers.As shown, no obvious change in emission wavelength of TO6C is caused immediately by the addition of S. aureus at 0 min (Figure 5F and Figure S29).After incubation for 5 min, the emission blue-shifts from 483 nm to about 480 nm and then almost remains unchanged over time (Figure 5F), suggesting the disassembly of TO6C aggregates into monomeric states.Accompanied with that, TO6C monomers penetrate into S. aureus with a high labeling rate of 100% (Figure 5E,G) and cause slight change in the surface zeta potential of S. aureus (Figure 5H).At this time, the staining efficiency of PI and the killing activity of TO6C reaches 98.6% and 100% at 5 min, respectively (Figure 5G and Figure S28, TO6C mission has no effect on the PI fluorescence signal (Figure S30)).This means, TO6C monomer shows the strong ability of destroying bacterial membrane due to its two long hexyl linkages, and can kill bacteria once penetrating into the bacterial membrane.As a result, with a relatively slower dynamics of entry into bacteria than TO2C and T2OE, TO6C still shows a fast dynamics of killing S. aureus and basically equilibrates within 5 min (Figure 5G).
These results reveal that the insertion dynamics for cationic amphiphile aggregates is inversely related to their stability.The less stable aggregates are more prone to disassemble into monomers upon contact with bacteria, which facilitates their insertion into bacterial membrane.But on the other hand, the dynamics of membrane destruction is positively associated with the ability of monomers to disrupt bacterial membranes.Increasing the hydrophobicity of cationic amphiphiles endows the monomers with strong membrane-disruption ability but also enhances the stability of aggregates.Consequently, both the stability of aggregates and the membrane-disruption ability of their monomers of cationic amphiphiles contribute to their dynamics of killing bacteria.

CONCLUSION
In conclusion, we developed three AIE-active cationic bolatype amphiphiles for dissecting the specific antimicrobial action and dynamics of cationic amphiphiles aggregates by obtaining the corresponding fluorescent information upon contact with bacteria.Based on the obvious change in emission wavelength upon disassembly, three amphiphiles can self-report their aggregated and monomeric state, thus capable of visually monitoring their interaction with bacteria.It has been demonstrated that three amphiphiles exert their antimicrobial activity through that their aggregates work as a monomer reservoir and disassemble into the monomers upon binding with bacteria, followed by penetrating into and disrupting the bacterial membrane.As for three amphiphiles, the hydrophilicity of monomers and stability of aggregates were finely controlled by tuning their linkers between two quaternary ammonium end groups and TPE skeleton.The stability of aggregates was confirmed to follow the order of TO6C > TO2C > T2OE.With the discrepancy in stability, TO2C, T2OE, and TO6C aggregates show different association tendency into monomers on contact with bacteria, which dominates the dynamics of their entry into bacteria.With the diverse hydrophobicity and the intrinsic difference of alkoxyl chain and alkyl chain in destroying membranes, the ability of monomers to disrupt bacterial membranes rank in the order of TO6C > TO2C > T2OE, which strongly affects the dynamics of membrane destruction.Thus, a reasonable balance between the stability of aggregates and membrane-disruption ability of monomers of cationic amphiphiles should be considered toward highly efficient antimicrobials.

F I G U R E 1
Chemical structure of three aggregation-induced emission (AIE)-active cationic amphiphiles and schematic illustration of the interaction of their aggregates with bacteria.F I G U R E 2 (A-C) Plots of fluorescence intensity and maximum wavelength of TO2C (A), T2OE (B) and TO6C (C) versus the concentration in phosphate buffered saline (PBS) solution, respectively.(D-F) Transmission electron microscopy (TEM) images and transmission electron diffraction (TED) patterns (insert) of TO2C (D), T2OE (E), and TO6C (F) at the concentration of 50 μM in PBS solution, respectively.(G and H) Size distribution and zeta potential results of three amphiphiles (50 μM) in PBS solution, respectively.Error bars: mean ± SD (n = 3).(I) Absorption and fluorescence spectra under the excitation of 340 nm of three amphiphiles (50 μM) in PBS solution.

F
I G U R E 4 (A) Fluorescence spectra of TO2C aggregates without and with the interaction of Staphylococcus aureus for different time in PBS solution, Ex: 340 nm.(B) The emission maximum of TO2C versus the incubation time with S. aureus.(C) confocal laser scanning microscopy (CLSM) images of S. aureus alone, T2OC aggregates and S. aureus after incubated with TO2C aggregates for the different time and then costained with 5 μg/mL of PI for 10 min, imaging conditions: Ex: 405 nm, Em: 450−600 nm for TO2C; Ex: 543 nm, Em: 600-700 nm for PI.(D) The plots of staining efficiency and killing activity of TO2C toward S. aureus at different incubation time and the staining efficiency of PI for S. aureus after treated by TO2C aggregates for the different time.Error bars: mean ± SD (n = 3).(E) Zeta potential results of S. aureus without and with TO2C aggregates, and TO2C aggregates themselves versus the time, respectively.[TO2C] = 100 μM.Error bars: mean ± SD (n = 3).

F
I G U R E 5 (A and E) CLSM images of T2OE and TO6C aggregates and Staphylococcus aureus after incubated with the aggregates for the different time and then costained by 5 μg/mL of PI for 10 min, respectively.Imaging conditions: Ex: 405 nm, Em: 450−600 nm for T2OE and TO6C; Ex: 543 nm, Em: 600-700 nm for PI.(B) and (F) The emission maximum of T2OE and TO6C versus the incubation time with S. aureus, respectively.(C and G) The plots of staining efficiency and killing activity of two amphiphiles toward S. aureus over time and the staining efficiency of PI for S. aureus after treated by the amphiphiles for different time.Error bars: mean ± (n = 3).(D and H) Zeta potential results of S. aureus without and with the aggregates, and the aggregates themselves versus the time, respectively.[T2OE] = 100 μM and [TO6C] = 50 μM.Error bars: mean ± SD (n = 3).