Skip to main content
NCBI home page
JACS Au logoLink to JACS Au
. 2025 Jan 22;5(2):675–683. doi: 10.1021/jacsau.4c00915

Unveiling the Role of Alkyl Chain in Boosting Antibacterial Selectivity and Cell Biocompatibility

Ziwei Deng †, Rongyuan Zhang †,●, Junyi Gong †, Zicong Zhang †, Lingyan Zhang ‡, Zijie Qiu †, Parvej Alam †, Jianquan Zhang †, Yong Liu §, Ying Li ∥, Zheng Zhao †,*, Ben Zhong Tang †,⊥,*
PMCID: PMC11862927  PMID: 40017763

Abstract

graphic file with name au4c00915_0009.jpg

Cationic amphiphiles have been demonstrated to be superior targeted antibacterial agents whose antibacterial activity exhibits a close relationship with their alkyl chain substituents. However, a systematic and deep investigation of the structure–property relationship is still pending. Meanwhile, cationic amphiphiles have a risk of accumulating in living mammalian cells, which poses a great threat to biosafety and clinical applications. In this study, a series of cationic amphiphilic aggregation-induced emission luminogens (AIEgens) with different alkyl chains (TPD-4, TPD-6, and TPD-12) have been developed with selective and variable antibacterial activity against Gram-positive bacteria depending on the alkyl chain length. Among them, TPD-6 with the intermediate alkyl chain length exhibited superior Gram-positive antibacterial performance. In addition, these cationic amphiphilic AIEgens had negligible invasiveness to mammalian cells. Molecular dynamics simulations revealed that the binding and deforming capabilities of the cationic amphiphilic AIEgens to the phospholipid bilayer of bacteria are responsible for their antibacterial activity. In vivo experiments indicated that TPD-6 also exhibited significant antibacterial and wound-healing abilities against Gram-positive bacteria.

Keywords: aggregation-induced emission, cationic amphiphiles, antibacterial selectivity, alkyl chain engineering, bacterial infection

Introduction

Bacterial infections have persisted throughout the history of human civilization, causing numerous life-threatening diseases such as the Black Death, phthisis, and typhoid fever.1−3 Since the discovery of penicillin in the 20th century, antibiotics have become an indispensable weapon in fighting against bacterial infection. However, the overuse of antibiotics aggravates drug-resistant bacterial occurrence and invalidates available antibiotics.4−6 In this regard, there is an urgent need for the development of efficient antibacterial agents to combat bacterial infections.7−9

Cationic amphiphiles with positive segments and hydrophobic units possess mimic-phospholipid structures and exhibit amphiphilic characteristics.10 These mimic-phospholipid structures enable their integration into bacterial membranes, thereby disrupting the bacterial morphology. Due to their distinct target site, which is unrelated to genetic substances, cationic amphiphiles present a reduced risk of genetic alteration and drug resistance, making them highly attractive in current antibacterial research.11,12 However, their poor selectivity often leads to the elimination of nonpathogenic bacteria alongside the intended pathogens. Some recent studies have underscored the crucial influence of the alkyl chain length of cationic amphiphiles on hydrophobic regulation at the atomic level, a factor closely linked to antibacterial selectivity.13−16 Yet, comprehensive investigations regarding the correlation between alkyl chain length and the specific antibacterial behavior remain limited. Furthermore, it is worthy to note that many reported cationic amphiphiles with long alkyl chains can be easily integrated into cell membranes and accumulate within living mammalian cells, posing significant biosafety concerns such as hemocompatibility.17 Consequently, the selective elimination of specific bacteria while minimizing the impact on mammalian cells has emerged as a pivotal challenge in the development of effective cationic amphiphiles.

Fluorescence imaging has been commonly employed as a visible technique for the mechanistic investigation of biological processes due to its inherent advantages in terms of convenience, high resolution, and real-time monitoring capabilities.18,19 Conventional luminogens mostly suffer from an aggregation-caused quenching effect, which limits their application in real-time biological process monitoring. In contrast, aggregation-induced emission luminogens (AIEgens) exhibit weak emission in dilute solutions but strong emission in aggregate or solid states, which enable a “light-up” feature of AIEgens when they accumulate in a biological environment.20−22 It is thus reasonable to integrate the advantages of AIEgens and cationic amphiphiles to afford a cationic amphiphilic AIEgen, which may provide a powerful tool to trace the interaction between antibacterial agents and bacteria and unveil the antibacterial mechanism with a visible approach.23−26

In this work, a series of AIEgens (TPD-4, TPD-6, and TPD-12) with varying alkyl chain lengths were designed and synthesized (Figure 1). Because long alkyl chains in the tail are likely to insert into the cell membrane and cause cytotoxicity, the AIEgens thus were designed with an alkyl chain bridge rather than a tail to depress their binding toward mammal cells.27 Subsequently, in vitro antibacterial experiments demonstrated their specificity in targeting Gram-positive bacteria (S. aureus and MRSA) rather than Gram-negative bacteria (E. coli). Notably, the distinct antibacterial activity against Gram-positive bacteria was modulated by the length of the alkyl chain. Moreover, these three compounds exhibited minimal invasiveness and negligible toxicity toward mammalian cells, as evidenced by fluorescence imaging and comprehensive biocompatibility assessments. Elucidating the mechanism that governs their preferential targeting of Gram-positive bacteria over Gram-negative bacteria and mammalian cells was accomplished through molecular dynamics (MD) simulations. This strategy of constructing AIEgens with different alkyl chain lengths has the potential to fill the map of the antibacterial agents.

Figure 1.

Figure 1

Open in a new tab

Molecule structures of AIEgens with different alkyl chains and proposed mechanism of the antibacterial activity.

Results and Discussion

Molecular Design and Photophysical Properties

Three AIEgens were designed with different alkyl chains, as shown in Figure 1. Since phospholipids are the main components of the bacterial cell membrane and typically feature hydrophilic head groups and hydrophobic tails, the three AIEgens were thus purposefully crafted with hydrophobic alkyl chains and two positively charged pyridine groups to mimic the amphipathic nature of phospholipids. We believe the similar amphipathic nature of cationic amphiphilic AIEgen as phospholipids may bolster their capability to interact with bacterial cell membranes. The insertion of the alkyl chain of AIEgens can alter the swelling states of bacterial cell membranes, leading to the disorganization of the lipid layer and thus killing the bacteria. However, the longer alkyl chain would cause a stronger cationic amphiphile–lipid layer interaction and a more severe disruption of the mammalian cell membrane.27 To decrease the cytotoxicity, the alkyl chain was designed in the middle of the molecular structure to reduce its exposure to the tails and minimize the interaction with the mammalian cell membrane. The AIEgens were named TPD-4, TPD-6, and TPD-12 depending on the alkyl chain length. Their chemical structures were well characterized by 1H NMR, 13C NMR, and HRMS (Figures S1–S9). In DMSO solutions, they displayed maximum absorptions of approximately 471, 478, and 477 nm, respectively, with high molar absorptivity (71,532, 78,118, and 75,404 M–1 cm–1) (Figures 2a and S10). Based on the density functional theory calculation of compounds TPD-4, TPD-6, and TPD-12, the absorption of intramolecular charge transfer states is attributed to the charge transfer from triphenylamine parts to pyridine units (Figure S11). Their maximum emission wavelengths in DMSO solution were found at around 742, 742, and 730 nm, respectively, showcasing near-infrared (NIR) emissions (Figure 2b). This NIR emission mitigates the interference of biological autofluorescence, enabling high-resolution bacterial imaging. The AIE character of the three molecules was demonstrated by measuring their PL spectra with the gradual addition of a poor solvent (ethyl acetate, EA) to the ethanol solution (Figures 2c,d and S12). These processes displayed increasing fluorescence intensity but blue-shifted emission with the addition of a poor solvent due to the formation of aggregates accelerating the restriction of molecular motion.

Figure 2.

Figure 2

Open in a new tab

(a) Normalized absorption spectra of TPD-4, TPD-6, and TPD-12 in a DMSO solution (5 μM). (b) Normalized photoluminescence (PL) spectra of TPD-4, TPD-6, and TPD-12 in a DMSO solution (20 μM). (c) PL spectra of TPD-4 in EA/ethanol mixtures with different EA fractions. (d) Plot of PL intensity (I/I0) of TPD-4, TPD-6, and TPD-12 versus different EA fractions, where I0 = PL intensity in pure ethanol. Eex = 460 nm (αAIE is I/I0).

In Vitro Antibacterial Assays

To explore the relationship between molecular structure and antibacterial activity, in vitro antibacterial experiments were conducted on E. coli (G–), S. aureus (G+), and MRSA (G+), (Figure 3a,b), which were selected as representatives for Gram-negative, Gram-positive, and drug-resistant Gram-positive bacteria, respectively. After incubation with TPD-4, TPD-6, and TPD-12, the bacterial viability of E. coli slightly decreased with the increased concentration of TPD-4, TPD-6, and TPD-12, suggesting the poor antibacterial ability of these compounds toward E. coli. In contrast, the antibacterial activities of TPD-4, TPD-6, and TPD-12 toward S. aureus are quite different. In detail, the bacterial viabilities of S. aureus treated by TPD-4, TPD-6, and TPD-12 were 20.4, 0.4, and 6.8% at 1 μM concentration and 77.1, 2.6, and 69.2% at an ultralow concentration of 0.25 μM. The results indicated that AIEgen TPD-6 was superior to TPD-4 and TPD-12 in antibacterial ability to S. aureus. For MRSA, the bacterial viabilities incubated with TPD-4, TPD-6, and TPD-12 were 102, 0, and 8.7%, respectively, at 1 μM concentration and 111.4, 2.5, and 75.7% at 0.25 μM concentration. These results showed that both TPD-6 and TPD-12 exhibit effective inhibition to the survival of S. aureus and MRSA rather than E. coli, demonstrating their strong antibacterial activity and selectivity toward Gram-positive bacteria. Given their similar structure and electrostatic potential, the length of alkyl chains was considered as the key factor to modulate their selectivity binding and antibacterial activity toward Gram-positive bacteria.

Figure 3.

Figure 3

Open in a new tab

Antibacterial activity of TPD-4, TPD-6, and TPD-12 toward E. coli (G–), S. aureus (G+), and MRSA (G+). (a) Photos of bacterial colonies and (b) relative bacterial viability incubated with TPD-4, TPD-6, and TPD-12 at different concentrations of 0.12, 0.25, 0.5, and 1 μM for 30 min.

Morphology Characterization

To further understand the antibacterial behavior of these cationic amphiphilic AIEgens, scanning electron microscopy (SEM) was employed to investigate the morphological changes of E. coli and S. aureus treated with AIEgens. After incubating with TPD-4, TPD-6, and TPD-12 at a concentration of 1 μM, it was found that the surfaces of the E. coli cell membrane remained smooth and intact as the control group, while the S. aureus cell membrane was prominently disrupted (Figure 4a). When decreasing the concentration of AIEgens to 0.5 μM, it was found that the cell membrane of S. aureus treated by TPD-6 represented a damaged surface, while the others kept their relative integrity similar to the one treated by PBS (Figure 4b). A detailed analysis showed that the cell membrane treated by TPA-6 was destructive, and the cytoplasm seemed to leak out (Figure 4b). It was thus concluded that these AIEgens killed S. aureus bacteria through damaging the cell membrane and their antibacterial activity relied on their capability to damage membranes. TPD-6 showed a better disruption to the Gram-positive bacterial cell membrane compared with TPD-4 and TPD-12, suggesting the six-carbon-length alkyl chains exhibit a stronger interaction with the bacterial cell membrane.

Figure 4.

Figure 4

Open in a new tab

(a) SEM images of E. coli and S. aureus incubated with TPD-4, TPD-6, and TPD-12 (1 μM) for 30 min. (b) SEM images and TEM images of S. aureus incubated with TPD-4, TPD-6, and TPD-12 (0.5 μM) for 30 min.

Fluorescence Imaging of AIEgens

To better understand how the cationic amphiphilic AIEgens interact with and kill bacteria, fluorescence imaging was employed as a visualization tool to study the interaction between AIEgens and bacteria through confocal laser scanning microscopy (CLSM). Herein, E. coli, MRSA, and S. aureus were stained by TPD-4, TPD-6, and TPD-12 and a commercial nucleic acid dye (Hoechst 33342). It was observed that there was a strong emission in S. aureus and MRSA, while no fluorescent signal was found in E. coli after incubation in the same condition (Figures 5a,b and S13). In addition, the fluorescence of AIEgens had less overlay with that of Hoechst 33342, suggesting that their anchored sites were related to the cell membrane of S. aureus and MRSA. Contrary to S. aureus and MRSA, no emission could be detected outside the blue fluorescence area of Hoechst 33342, suggesting that AIEgens weakly bound with E. coli’s cell membrane. It is noteworthy that the enhanced emission after AIEgens incubated with the bacteria implied that the AIEgens gradually accumulated and aggregated in the bacterial cell membrane. Moreover, the PL intensities of AIEgens incubated with S. aureus were generally stronger than those incubated with E. coli, which further supported the fact that AIEgens could much more easily interact and aggregate in the S. aureus bacteria cell membrane (Figure S14). The AIEgens’ emission exhibited a slight blue shift after incubation with bacteria possibly because AIEgens interacted with bacterial cell membranes, which stabilized their rigid molecular conformation.

Figure 5.

Figure 5

Open in a new tab

CLSM images of (a) S. aureus and (b) E. coli incubated with TPD-4, TPD-6, and TPD-12 (0.5 μM) for 20 min and incubated with Hoechst 33342 (5 μg mL–1) for 15 min. Zeta potentials of (c) S. aureus and (d) E. coli incubated with TPD-4, TPD-6, and TPD-12 (0.5 μM) for 30 min. Membrane depolarization results of (e) S. aureus and (f) E. coli incubated with TPD-4, TPD-6, and TPD-12.

Surface zeta potentials of bacteria incubated with AIEgens were then conducted. Without AIEgens treatment, the surface zeta potentials of E. coli and S. aureus were measured to be −29.6 and −25.0 mV (Figure 5c,d), respectively. After incubation, the zeta potential was not changed in terms of E. coli but sharply increased for S. aureus. Due to their similar charge carriers (Figure S15), these results indicated that AIEgens have a stronger interaction with S. aureus than E. coli, which could affect their antibacterial selectivity.

In particular, surface potentials of TPD-6-incubated S. aureus showed a more obvious change than TPD-4 and TPD-12, which was consistent with their antibacterial activity. Furthermore, the cell membrane depolarization was detected by a commercial fluorescent dye, 3,3′-dipropylthiadicarbocyanine iodide (DiSC35), which is sensitive to the electrostatic potential of the cell membrane. After incubation with TPD-4, TPD-6, and TPD-12, the fluorescence intensity of DiSC35 gradually increased in the case of S. aureus, while the intensity remained stable in the case of E. coli (Figure 5e,f). This indicated that the AIEgens showed a better combination with the S. aureus cell membrane, especially for TPD-6, which had the best binding ability to S. aureus.

Cell Biocompatibility of AIEgens

The biosafety of antibacterial agents to mammalian cells is quite significant, and fluorescence bioimaging thus was conducted by CLSM to reveal the interaction between AIEgens and mammalian cells. Here, we chose the HeLa cell, a kind of human cervical cancer cell line, as a model due to its active metabolism, which is suitable for exploring the living cell invasiveness of extracellular molecules. After incubation with AIEgens at an antibacterial concentration of 0.5 μM, a negligible fluorescence signal from living HeLa cells was observed (Figure 6a). This result indicated that AIEgens had weak binding and little invasiveness to HeLa cells. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, a standard method to evaluate cell toxicity, showed that these AIEgens had low toxicity (Figure 6b) to HeLa cells even with the concentration increasing. To further prove the biocompatibility, the NIH/3T3 line, a fibroblast cell, was employed for verification. As depicted in Figure 6c, after incubation with AIEgens, NIH/3T3 cells exhibited a weak intracellular accumulation. The MTT assay proved that AIEgens had negligible toxicity to NIH/3T3 cells (Figure 6d). These results demonstrated the superior biocompatibility of cationic amphiphilic AIEgens with mammalian cells.

Figure 6.

Figure 6

Open in a new tab

CLSM images of (a) HeLa and (c) NIH/3T3 cells after incubation with TPD-4, TPD-6, and TPD-12 (0.5 μM) for 30 min. Cell viability of (b) HeLa and (d) NIH/3T3 cells after incubation with different concentrations of TPD-4, TPD-6, and TPD-12 for 30 min.

MD Simulation

To gain deep insights into the interaction process of cationic amphiphilic AIEgens with bacteria and mammalian cells, MD simulation was carried out on TPD-4, TPD-6, and TPD-12. Three kinds of lipid combinations—POPE/POPG (1:3), POPE/POPG (3:1), and POPC—were utilized as models to mimic the structures of Gram-positive bacterial phospholipids, Gram-negative bacterial phospholipids, and mammalian cell phospholipids, respectively (Figure S16).28,29 It was proposed that the process of membrane disruption consisted of three parts, adsorption, electrostatic interaction, and penetration, which gradually cause membrane disruption according to previous research.27 Herein, the absorption process from an aqueous solution into the lipid layers was simulated. The AIEgens exhibited an affinity with the peripheral hydrophilic end group of the phospholipid bilayer (Figures S17 and S18). With the simulation time increasing, TPD-4, TPD-6, and TPD-12 absorbed on the surface of the phospholipid’s bilayer gradually entered and assembled within the phospholipid’s bilayer (Figures S17 and S18). To evaluate the driving force of the absorption process, the interaction energy including electrostatic energy and van der Waals energy were analyzed. The interaction energy between AIEgens and POPE/POPG (1:3) was superior to that between AIEgens and the POPE/POPG (3:1) bilayer as well as the POPC bilayer, reflecting a better interaction between POPE/POPG (1:3) bilayers and AIEgens (Figures 7a and S19). Among the three cationic amphiphilic AIEgens, TPD-6 exhibited a higher interaction energy than TPD-4 and TPD-12, which suggested it had stronger binding with the POPE/POPG (1:3) bilayers.

Figure 7.

Figure 7

Open in a new tab

MD simulation to investigate the specific mechanisms of AIEgens (TPD-4, TPD-6, and TPD-12) against bacterial membranes. (a) Calculated interaction energy between AIEgens and the phospholipid bilayer. (b) Sequential snapshots of MD simulations of interactions between AIEgens and the phospholipid bilayer. (c) MSD of AIEgens inserted in a phospholipid bilayer. (d–g) Local Scd of the POPE/POPG bilayer corresponding to sn1 chains and sn2 chains. (h) Schematic illustration of the process of AIEgens’ penetration into a membrane.

To understand how the AIEgens affect the phospholipid bilayer, we conducted a simulation where TPD-4, TPD-6, and TPD-12 were inserted into the inner layer of POPE/POPG (1:3) bilayers (Figure 7b). The mean square displacement (MSD), reflecting the movement of AIEgens inside the lipid layers, was calculated. As shown in Figure 7c, among the three AIEgens, TPD-6 showed the most significant increase in the MSD, indicating that TPD-6 displayed the greatest fluctuation inside the bilayer compared to the others and therefore strongly disturbed the bilayer membrane structure. In contrast, the MSD of TPD-4 exhibited the smallest increase, suggesting its weakest capability of fluctuation and membrane disturbance. The simulation results were well consistent with the antibacterial performance of the three AIEgens.

To explore how the AIEgens affect the molecular arrangement of phospholipids inside the bilayers, the deuterium order parameter (Scd) was calculated, which stands for the order of lipid chain arrangement and reflects the membrane order as well as structural integrity. Lower Scd usually means larger membrane disorganization.30 The Scd of POPE/POPG (1:3) bilayers in the presence of AIEgens was calculated by the continuous lipid configuration extracted from a 200 ns MD simulation (Figure 7b). TPD-6 showed a lower Scd value of POPE (along the heptadecanoyl (sn1) and (Z)-9-octadecenoyl (sn2) of POPE) and POPG (along the heptadecanoyl (sn1) and (Z)-9-octadecenoyl (sn2) of POPG) (Figure S16) compared with TPD-4 and TPD-12, indicating that TPD-6 possessed the strongest perturbance toward the phospholipid bilayer, while TPD-4 and TPD-12 had a weak effect on the phospholipid order (Figure 7d–g). The MD simulation gave an insight into the permeating process of AIEgens into the membrane. They first absorbed on the membrane surface and then had a specifically strong interaction with a certain membrane (POPE/POPG (1:3) lipid bilayer), finally penetrating into the membrane and causing membrane disorganization and disruption (Figure 7h).

Evaluation of the In Vivo Infection Model

An in vivo experiment utilizing a wound infection model was performed to evaluate the antibacterial activity of these cationic amphiphilic AIEgens. A wound with a size of about 0.7 cm × 0.7 cm was created on the back of BALB/c mice and then infected by MRSA. The infected mice were divided into four groups, which were treated with PBS, TPD-4, TPD-6, and TPD-12, separately, as depicted in Figure 8a. The area of the wound was photographed, and the relative body weight was recorded on day 1, day 2, day 4, and day 6 (Figures 8b and S20). The statistical analysis showed the healing changes of wounds treated with PBS, TPD-4, TPD-6, and TPD-12 with time (Figure 8c). Among these, the TPD-6-treated wound exhibited a faster healing process with the smallest area of the wound on day 6 (Figure S21). Moreover, MRSA collected from the wound skin treated with TPD-6 showed a sharply reduced number as observed by bacteria colony counting (Figures 8d and S22). The H&E-stained slices of wound tissues treated with TPD-6 on day 6 revealed the presence of newborn blood vessels and hair follicles (Figure 8e), features that were absent in the slices treated with PBS, TPD-4, and TPD-12. The result from immunohistochemistry (IHC) slices on day 6 (Figure 8f) showed a higher expression level of VEGF in TPD-6-treated infected mice compared with the PBS group, TPD-4 group, and TPD-12 group, further confirming the fast generation of new blood vessels. Inflammation is a key factor reflecting the wound infection. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) play an important role in immune responses. From the results of IHC slices on day 6 (Figure 8g,h), a lower expression level of TNF-α and IL-6 was observed in TPD-6-treated infected mice among the four treatment groups. In addition, there was no pathological change observed in the H&E-stained slices of the five major organs (heart, liver, spleen, lung, and kidney) from four groups (Figure S23). The hemolysis ratios were all below the permissible limit (5%) as observed from the hemolytic test (Figure S24). These results indicated their good biocompatibility and demonstrated their potential in the treatment of bacterial infections.

Figure 8.

Figure 8

Open in a new tab

(a) Procedure illustration of the establishment and treatment of the skin wound infection model. (b) Photographs and (c) relative wound area changes of MRSA-infected mice during the wound-healing process after different treatments. (d) Statistical analysis of bacterial colonies collected from wound tissue on the sixth day. (e) H&E staining and (f)–(h) IHC staining (VEGF, IL-6, and TNF-α factors) slices of wound tissues collected on the sixth day. Error bar: mean ± SD (n = 3, *P < 0.05, **P < 0.01).

Conclusions

In summary, a series of cationic amphiphilic AIEgens (TPD-4, TPD-6, and TPD-12) with different lengths of alkyl chains were developed to investigate the relationship between structure and specific antibacterial performance as well as their biocompatibility. In vitro antibacterial experiments indicated that the cationic amphiphilic AIEgens generally showed antibacterial specificity to Gram-positive bacteria, and AIEgens with different lengths of alkyl chains exhibited different Gram-positive antibacterial activities. The distinct capacities of these AIEgens for disrupting bacterial cell membranes were elucidated through SEM and TEM, where TPD-6 exhibited a superior membrane-disrupting ability in comparison to those of both TPD-4 and TPD-12. Fluorescence bioimaging effectively visualized the process of AIEgens interacting with bacteria. Zeta potential analysis and membrane depolarization measurements confirmed that there is a stronger interaction between TPD-6 and Gram-positive bacteria. In addition, these cationic amphiphilic AIEgens exhibited low invasiveness and toxicity toward mammalian cells. MD simulation disclosed the interaction between AIEgens and the simulated phospholipid bilayers of Gram-positive bacteria, showing that TPD-6 exhibited a stronger perturbance and disorganization of the phospholipid chain, which is responsible and crucial for its superior antibacterial activity. The in vivo results also confirmed the superior antibacterial activity of TPD-6. This work not only provided an effective strategy for designing antibacterial agents but also fundamentally unveiled the antibacterial mechanism of these cationic amphiphilic AIEgens, which is of great importance for the development of new antibacterial agents.

Methods

Antibacterial Assay In Vitro

Bacteria (E. coli, S. aureus, and MRSA) in a liquid LB medium were washed in PBS three times. The OD value of the collected suspension was OD600 = 1.0. They were diluted in PBS (1 × 106 folds) and then incubated with AIEgens (TPD-4, TPD-6, and TPD-12) for 30 min. The samples were diluted further in PBS (1 × 104 folds), and 50 μL of suspension was spread on solid LB plates. After incubation for 18 h at 37 °C, the bacteria on plates were counted and the photographs were captured by a digital camera. The calculation of bacterial viability was based on the equation of B/A* 100% (A stands for the mean number of bacterial colonies without treatment and B stands for the mean number of bacterial colonies after treatment with AIEgens).

Surface Morphology Observation of the Bacterial Assay

Bacteria (E. coli and S. aureus) in a liquid LB medium were washed in PBS three times. Then the bacterial suspension was diluted in PBS (OD600 = 0.1). After incubation with AIEgens for 30 min at 37 °C, the bacteria were collected at 8000 rpm for 2 min. Then they were resuspended by 50 μL of PBS, spread in silicon slides, and transferred in a 4% paraformaldehyde solution at 4 °C overnight. The treated bacteria were dehydrated by a graded ethanol solution from 10 to 100% and further dried in a vacuum drying oven for 2 h for SEM analysis. Bacteria (S. aureus) were washed in PBS three times, and the bacterial suspension was diluted in PBS (OD600 = 0.1). After incubation with AIEgens for 30 min at 37 °C, the bacteria were collected at 8000 rpm for 2 min, and then they were fixed with 2.5% glutaraldehyde at 4 °C overnight for TEM analysis.

Surface Zeta Potential of Bacteria

The bacteria (E. coli and S. aureus) (OD600 = 0.1) were washed in PBS three times. After incubation with AIEgens for 30 min at 37 °C, the samples were collected at 8000 rpm for 2 min and resuspended in 1 mL of deionized water for zeta potential measurements. The bacteria under the same condition without treatment were the same as the control sample.

Membrane Depolarization

E. coli and S. aureus were washed with a HEPES buffer (5 mM, pH 7.4, containing 20 mM glucose). Following washing, the bacteria were resuspended in the same buffer (OD600 = 0.1) and then were incubated with 10 μM DiSC35 for 1 h at 37 °C. After that, 100 mM KCl was added to equilibrate the cytoplasmic and external K+. Finally, the bacterial suspensions were treated with three different molecules, and the fluorescence intensity of DiSC35 was monitored regularly for 50 min using a microplate reader. The excitation wavelength was set to 622 nm, and the emission wavelength was defined as 670 nm. Bacterial suspensions without treatment of the three molecules were used as negative controls.

CLSM Observation

The bacteria (E. coli, S. aureus, and MRSA) (OD600 = 0.1) were washed in PBS three times. After incubation with AIEgens for 30 min at 37 °C, the samples were collected at 8000 rpm for 2 min and resuspended in 50 μL of PBS. Then they were stained with Hoechst 33345 (5 μg/mL) for 10 min at ambient temperature. The treated bacterial suspension was spread onto a glass slide and then was covered with a coverslip for CLSM imaging. The mammalian cells (HeLa and NIH/3T3) were incubated with AIEgens for 30 min at 37 °C and then washed in PBS three times for CLSM imaging. The excited wavelength of TPD-4, TPD-6, and TPD-12 was 488 nm, and the emissive wavelengths were collected from 650 to 750 nm.

Acknowledgments

This research work was financially supported by NSFC (52273197), the Shenzhen Key Laboratory of Functional Aggregate Materials (ZDSYS20211021111400001), the Science and Technology Plan of Shenzhen (JCYJ2021324134613038, KQTD20210811090142053, JCYJ20220818103007014, GJHZ20210705141810031), and the Innovation and Technology Commission (ITC—CNERC14SC01). The authors would like to thank the Materials Characterization and Preparation Center, The Chinese University of Hong Kong, Shenzhen, for NMR and HRMS measurements. The authors are also grateful to Ms. Xuan Li and Ms. Guimiao Liu for their help with HRMS measurement. The authors acknowledge Miss. Yanling Liu and Mr. Haifei Wen for their help.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.4c00915.

  • Experimental procedures; theoretical calculation and MD simulation details; copies of 1H NMR spectra, 13C NMR spectra, and HRMS data; UV–vis spectra; PL spectra; zeta potential data; CLSM images; statistics analysis; and images collected from animal experiments (PDF)

Author Contributions

# Z.D. and R.Z. contributed equally to this paper.

The authors declare no competing financial interest.

Special Issue

Published as part of JACS Auspecial issue “Advances in Small Molecule Activation Towards Sustainable Chemical Transformations”.

Supplementary Material

au4c00915_si_001.pdf (3.2MB, pdf)

References

  1. Schuenemann V. J.; Bos K.; DeWitte S.; Schmedes S.; Jamieson J.; Mittnik A.; Forrest S.; Coombes B. K.; Wood J. W.; Earn D. J.; White W.; Krause J.; Poinar H. N. Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid of Yersinia pestis from victims of the Black Death. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, E746–E752. 10.1073/pnas.1105107108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Stead W. W.; Senner J. W.; Reddick W. T.; Lofgren J. P. Racial differences in susceptibility to infection by Mycobacterium tuberculosis. N. Engl. J. Med. 1990, 322, 422–427. 10.1056/NEJM199002153220702. [DOI] [PubMed] [Google Scholar]
  3. Connor B. A.; Schwartz E. Typhoid and paratyphoid fever in travellers. Lancet Infect. Dis. 2005, 5, 623–628. 10.1016/S1473-3099(05)70239-5. [DOI] [PubMed] [Google Scholar]
  4. Hamad B. The antibiotics market. Nat. Rev. Drug Discovery 2010, 9, 675. 10.1038/nrd3267. [DOI] [PubMed] [Google Scholar]
  5. Kollef M. H. Broad-spectrum antimicrobials and the treatment of serious bacterial infections: getting it right up front. Clin. Infect. Dis. 2008, 47, S3–S13. 10.1086/590061. [DOI] [PubMed] [Google Scholar]
  6. Li X.; Bai H.; Yang Y.; Yoon J.; Wang S.; Zhang X. Supramolecular antibacterial materials for combatting antibiotic resistance. Adv. Mater. 2019, 31, 1805092 10.1002/adma.201805092. [DOI] [PubMed] [Google Scholar]
  7. Zhou K.; Wang S.; Xu L.; Li H.; Wang Y.; Qiu Z.; Zhang G.; Zhao Z.; Tang B. Z. AIEgen-based smart system for fungal-infected wound monitoring and on-demand photodynamic therapy. Matter 2023, 6, 3449–3462. 10.1016/j.matt.2023.06.028. [DOI] [Google Scholar]
  8. Zhang Z.; Deng Z.; Zhu L.; Zeng J.; Cai X. M.; Qiu Z.; Zhao Z.; Tang B. Z. Aggregation-induced emission biomaterials for anti-pathogen medical applications: detecting, imaging and killing. Regener. Biomater. 2023, 10, rbad044 10.1093/rb/rbad044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ye Z.; He W.; Zhang Z.; Qiu Z.; Zhao Z.; Tang B. Z. AIEgens for microorganism-related visualization and therapy. Interdiscip. Med. 2023, 1, e20220011 10.1002/INMD.20220011. [DOI] [Google Scholar]
  10. Tiller J. C.; Liao C.-J.; Lewis K.; Klibanov A. M. Designing surfaces that kill bacteria on contact. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 5981–5985. 10.1073/pnas.111143098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Yang Z.; Huang R.; Zheng B.; Guo W.; Li C.; He W.; Wei Y.; Du Y.; Wang H.; Wu D.; Wang H. Highly stretchable, adhesive, biocompatible, and antibacterial hydrogel dressings for wound healing. Adv. Sci. 2021, 8, 2003627 10.1002/advs.202003627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. He P.; Lv F.; Liu L.; Wang S. Cationic conjugated polymers for detection and inactivation of pathogens. Sci. China: Chem. 2017, 60, 1567–1574. 10.1007/s11426-017-9185-8. [DOI] [Google Scholar]
  13. Wang J.; Li J.; Shen Z.; Wang D.; Tang B. Z. Phospholipid-Mimetic Aggregation-Induced Emission Luminogens for Specific Elimination of Gram-Positive and Gram-Negative Bacteria. ACS Nano 2023, 17, 4239–4249. 10.1021/acsnano.2c05821. [DOI] [PubMed] [Google Scholar]
  14. Ji Y.; Li J.; Chen C.; Piao C.; Zhou X.; Yoon J. Wash-Free Bacterial Gram-Typing and Photodynamic Inactivation with Long-Chain-Tailed BODIPY Derivatives. Biomater. Res. 2024, 28, 0069 10.34133/bmr.0069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim H.; Lee Y. R.; Jeong H.; Lee J.; Wu X.; Li H.; Yoon J. Photodynamic and photothermal therapies for bacterial infection treatment. Smart Mol. 2023, 1, e20220010 10.1002/smo.20220010. [DOI] [Google Scholar]
  16. Li H.; Zhang L.; Han J.; Kim D.; Kim H.; Ha J.; Wang J.; Yoon J. Molecular structural evolution of near-infrared cationic aggregation-induced emission luminogens: preclinical antimicrobial pathogens activities and tissues regeneration. CCS Chem. 2022, 4, 487–500. 10.31635/ccschem.021.202101206. [DOI] [Google Scholar]
  17. Zheng L.; Zhu Y.; Sun Y.; Xia S.; Duan S.; Yu B.; Li J.; Xu F. J. Flexible Modulation of Cellular Activities with Cationic Photosensitizers: Insights of Alkyl Chain Length on Reactive Oxygen Species Antimicrobial Mechanisms. Adv. Mater. 2023, 35, 2302943 10.1002/adma.202302943. [DOI] [PubMed] [Google Scholar]
  18. Chinen A. B.; Guan C. M.; Ferrer J. R.; Barnaby S. N.; Merkel T. J.; Mirkin C. A. Nanoparticle probes for the detection of cancer biomarkers, cells, and tissues by fluorescence. Chem. Rev. 2015, 115, 10530–10574. 10.1021/acs.chemrev.5b00321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Roy E.; Nagar A.; Chaudhary S.; Pal S. AIEgen-Based Fluorescent Nanomaterials for Bacterial Detection and its Inhibition. ChemistrySelect 2020, 5, 722–735. 10.1002/slct.201904092. [DOI] [Google Scholar]
  20. Weiss J. Fluorescence of organic molecules. Nature 1943, 152, 176–178. 10.1038/152176a0. [DOI] [Google Scholar]
  21. Luo J.; Xie Z.; Lam J. W.; Cheng L.; Chen H.; Qiu C.; Kwok H. S.; Zhan X.; Liu Y.; Zhu D.; Tang B. Z. Aggregation-induced emission of 1-methyl-1, 2, 3, 4, 5-pentaphenylsilole. Chem. Commun. 2001, 18, 1740–1741. 10.1039/B105159H. [DOI] [PubMed] [Google Scholar]
  22. Gu H.; Sun W.; Du J.; Fan J.; Peng X. Dual-acceptor engineering of donor-acceptor type molecules for all-round boosting anti-tumor phototherapy. Smart Molecules 2023, 1, e20230014 10.1002/smo.20230014. [DOI] [Google Scholar]
  23. Wang D.; Tang B. Z. Aggregation-induced emission luminogens for activity-based sensing. Acc. Chem. Res. 2019, 52, 2559–2570. 10.1021/acs.accounts.9b00305. [DOI] [PubMed] [Google Scholar]
  24. Kang M.; Zhang Z.; Song N.; Li M.; Sun P.; Chen X.; Wang D.; Tang B. Z. Aggregation-enhanced theranostics: AIE sparkles in biomedical field. Aggregate 2020, 1, 80–106. 10.1002/agt2.7. [DOI] [Google Scholar]
  25. Qi J.; Ou H.; Liu Q.; Ding D. Gathering brings strength: How organic aggregates boost disease phototheranostics. Aggregate 2021, 2, 95–113. 10.1002/agt2.25. [DOI] [Google Scholar]
  26. Zhou C.; Ding Z.; Guan W.; Tang B. Z. Self-reporting fluorescence deciphers the antibacterial nature of cationic amphiphiles: Monomer or aggregate?. Aggregate 2023, 4, e366 10.1002/agt2.366. [DOI] [Google Scholar]
  27. Ding X.; Duan S.; Ding X.; Liu R.; Xu F. J. Versatile antibacterial materials: an emerging arsenal for combatting bacterial pathogens. Adv. Funct. Mater. 2018, 28, 1802140 10.1002/adfm.201802140. [DOI] [Google Scholar]
  28. Lee J.; Jung S. W.; Cho A. E. Molecular insights into the adsorption mechanism of human β-defensin-3 on bacterial membranes. Langmuir 2016, 32, 1782–1790. 10.1021/acs.langmuir.5b04113. [DOI] [PubMed] [Google Scholar]
  29. Denning E. J.; Beckstein O. Influence of lipids on protein-mediated transmembrane transport. Chem. Phys. Lipids 2013, 169, 57–71. 10.1016/j.chemphyslip.2013.02.007. [DOI] [PubMed] [Google Scholar]
  30. Khalid S.; Piggot T. J.; Samsudin F. Atomistic and coarse grain simulations of the cell envelope of Gram-negative bacteria: what have we learned?. Acc. Chem. Res. 2018, 5, 180–188. 10.1021/acs.accounts.8b00377. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

au4c00915_si_001.pdf (3.2MB, pdf)

Articles from JACS Au are provided here courtesy of American Chemical Society

RESOURCES