Molecular Structural Evolution of Near-Infrared Cationic Aggregation-Induced Emission Luminogens: Preclinical Antimicrobial Pathogens Activities and Tissues Regeneration

Open AccessCCS ChemistryRESEARCH ARTICLE1 Feb 2022Molecular Structural Evolution of Near-Infrared Cationic Aggregation-Induced Emission Luminogens: Preclinical Antimicrobial Pathogens Activities and Tissues Regeneration Haidong Li†, Liuwei Zhang†, Jingjing Han†, Dayeh Kim†, Heejeong Kim, Jeongsun Ha, Jingyun Wang and Juyoung Yoon Haidong Li† Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760 , Liuwei Zhang† State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024 School of Bioengineering, Dalian University of Technology, Dalian 116024 , Jingjing Han† Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760 , Dayeh Kim† Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760 , Heejeong Kim Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760 , Jeongsun Ha Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760 , Jingyun Wang *Corresponding authors: E-mail Address: email protected E-mail Address: email protected State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024 School of Bioengineering, Dalian University of Technology, Dalian 116024 and Juyoung Yoon *Corresponding authors: E-mail Address: email protected E-mail Address: email protected Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760 https://doi.org/10.31635/ccschem.021.202101206 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Increasingly infectious diseases from microbial pathogens (including bacteria and fungi) threaten human health: a situation that has aroused public health concern around the world. Unfortunately, broad-spectrum antimicrobial agents for treatment resistance pathogens and molecular research on their antimicrobial mechanisms are still scarce. Thus, the development of smart agents against microbial infection for surmounting the above dilemmas is an urgent task. In this contribution, we have tactfully designed a family of flexible aggregation-induced emission luminogens (AIEgens) with various alkyl chain lengths and successfully optimized a cationic AIEgen TPA-S-C6-NMe3 + based on the molecular relay strategy for killing both bacteria and fungi in vitro with desired results under white light irradiation, superior to traditional commercial photosensitizers including methylene blue, chlorin e6, and protoporphyrin IX. The cationic AIEgen TPA-S-C6-NMe3 + was bound to microbial pathogens via electrostatic and hydrophobic forces and exerted antimicrobial efficacy due to the synergistic effect of alkyl chain length, reactive oxygen species (ROS) generation capability, and two positive charges. Remarkably, AIEgen TPA-S-C6-NMe3 + also exhibited a striking antimicrobial activity in vivo, and promoted the generation of new blood vessels and fibroblasts in bacteria-infected tissues, which was beneficial for wound healing in mice. Overall, we expect that our work could provide a powerful tool against microbial pathogens to avoid infections and to promote tissues regeneration in clinical practice. Download figure Download PowerPoint Introduction Microbial pathogens exist everywhere and are intimately related to all aspects of human life (including food, water, the environment, and so on).1–5 Bacteria (Gram positive and Gram negative) and fungi are the main causes of microbial pathogenic infections, resulting in extremely severe consequences in clinical practice, such as fatal diseases and increasing patient mortality.6–12 For instance, pathogenic bacteria cause common diseases (including dermatosis, bacteremia, toxemia, sepsis, and tuberculosis) and are linked to cancer through two mechanisms: production of carcinogenic bacterial metabolites and induction of chronic inflammation.13–18 The fungal pathogen Candida albicans is a main cause of morbidity and mortality in immunocompromised patients and is also responsible for hospital-acquired infections due to the formation of fungal biofilms on implanted medical devices.19,20 In clinical practice, multimicrobial infections resulting from multiple pathogens are prevalent among patients.21 Antibiotics became important tools for saving the lives of countless infected people as soon as they were discovered.22 Unfortunately, as microbial pathogens become more resistant to antibiotics, these weapons are gradually losing their power.23,24 Additionally, there are few reports of highly effective broad-spectrum antimicrobial agents. The above dilemmas have inspired us to develop reliable agents for fast labeling and treatment of microbial pathogens without causing antimicrobial resistance. Structurally, the significant difference between bacteria and fungi is the chemical composition of their cell walls.25 Gram-positive bacteria have a thick layer of cross-linked peptidoglycan outside the cytoplasmic membrane, which results in porosity of the cell walls. Teichoic acids, with negative charge, intercalate the thick peptidoglycan layer to make Gram-positive bacteria negatively charged. For Gram-negative bacteria, the cell wall consists of an outer member with lipopolysaccharides (LPSs) and a relatively thin cross-linked peptidoglycan network. In contrast, fungi have a highly organized cell wall structure that is composed of mannoproteins, β-glucans, and chitin. Interestingly, surfaces of bacteria and fungi are both negatively charged. Based on this feature, antimicrobial agents are commonly designed to bear cationic units to regulate the electrostatic interactions between agents and bacteria, which can be used to efficiently kill microbial pathogens.26–28 Fluorescence imaging has received much attention in the detection of bacteria and fungi because of its outstanding advantages, such as high sensitivity, noninvasiveness, fast responsiveness, and simple operation.29–34 Moreover, photodynamic therapy (PDT) has emerged as a promising method for the clinical treatment of microbial infections, mainly because photodynamic antibiotics do not cause antimicrobial resistance.26,35 Unfortunately, conventional photosensitizers (PSs), such as phthalocyanine and porphyrin, are prone to the aggregation of π–π stacking due to their large rigid planar structures, leading to fluorescence quenching and reduced production of reactive oxygen species (ROS) under light irradiation, which seriously impedes their practical applications.36 The emergence of aggregation-induced emission luminogens (AIEgens) provides a powerful solution to the aforementioned issues.37,38 In contrast to the aggregation-caused quenching (ACQ) effect, AIEgens have almost no fluorescence in a good solution, but emit intense fluorescence in a poor solution due to the formation of aggregates that restrict intramolecular free motions.39–42 Considering their outstanding characteristics, including high photostability, large Stokes shift, satisfactory quantum yield, and ideal reactive oxygen production capacity in aggregates, AIEgens have been widely employed for fluorescence imaging in cells and in vivo and are used in PDT of bacteria and tumors.43–50 Additionally, the increasing popularity of smart AIEgens is attributed to their intrinsic ability to overcome undesirable aggregation prior to the attainment of the target,51 especially for amphiphilic AIE PSs that label microbial pathogens with a superior signal-to-noise ratio. As we know, considerable efforts have been made to develop novel AIE PSs for antibacterial activities with satisfactory effects in recent years. However, amphiphilic AIEgens with prominent efficacy against broad-spectrum bacteria and fungi are still very scarce. Additionally, systematic molecular studies of AIE agents for improving the antibacterial mechanism are also lacking, which is likely to be an important guideline for the rational development of next-generation advanced antimicrobial agents. In this contribution, based on the strategy of a molecular relay, a smart cationic AIEgen TPA-S-C6-NMe3 + with amphiphilic and near-infrared (NIR) emission characteristics, has been rationally designed and developed for efficiently dealing with antimicrobial pathogens to surmount the elusive goal. As depicted in Scheme 1, by changing the alkyl chain length at the end of the frame structure TPA-S-C0, the AIEgen TPA-S-C6 was optimized to have the best inhibitory effect on Staphylococcus aureus (Gram positive) and Escherichia coli (Gram negative). However, AIEgen TPA-S-C6 exhibited no obvious photodynamic antibacterial effect on the antibiotic-resistant bacterial strain extended spectrum beta-lactamase (ESBL) E. coli. Therefore, another positive charge was added to the tail to construct the more powerful amphiphilic AIEgen TPA-S-C6-NMe3 +, which could be used to kill common microbial pathogens including S. aureus, E. coli, ESBL E. coli, and even C. albicans (fungi) by the synergistic effect of suitable alkyl chain length, striking 1O2 generation capability, and two positive charges, making it superior to traditional commercial PSs including methylene blue (MB), chlorin e6 (Ce6), and protoporphyrin IX (PpIX). Additionally, its antimicrobial mechanism was revealed by confocal fluorescence imaging, fluorescence spectroscopy, and scanning electron microscopy (SEM). More importantly, amphiphilic AIEgen TPA-S-C6-NMe3 + was also employed for photodynamic antibacterial application in vivo with satisfactory results. All compounds were facilely synthesized according to the preparation method shown in Supporting Information Figure S1 and were fully characterized by nuclear magnetic resonance and high-resolution mass spectrometry ( Supporting Information Figures S2–S40). Scheme 1 | Schematic illustration of molecular structural evolution of the NIR cationic AIEgen TPA-S-C6-NMe3 + for efficient broad-spectrum antimicrobial activity and the promotion of tissue generation. Download figure Download PowerPoint Experimental Methods Materials and instrumentation Nuclear magnetic resonance (1H and 13C NMR) spectra were obtained using a Bruker 300 MHz spectrometer (Germany). Electrospray ionization high-resolution mass spectrometry (ESI-HRMS) was carried out on a Synapt G2 high definition mass spectrometer (Waters, Manchester, U.K.), which was operated on MassLynx 4.1 software at the Korea Basic Science Institute (KBSI, Ochang, Center of Research Equipment). Both fluorescence and UV–vis spectra were obtained using a 1 cm optical path length cell at room temperature. Fluorescence spectra were gathered using a FluroMate FS-2 Spectrometer (Scinco). UV–vis absorption spectroscopy measurements were performed on an EVOLUTION 201 (Thermo scientific, America). Confocal images of bacteria were acquired using a LSM 780 NLO (Carl Zeiss, Germany). Confocal images of fungi were acquired using a FV3000 confocal microscope (Olympus, Japan). SEM images were acquired on a JSM-6700F (JEOL, Japan) operating at an acceleration voltage of 10.0 kV. Nano-ZS (Mervern, Britain) was used for dynamic light scattering (DLS) and zeta (ζ) potential measurements. Singlet oxygen detection 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA; 50 μM) was employed to detect singlet oxygen generation of Ce6 (10 μM), rose bengal (RB; 10 μM), TPA-S-C0 (10 μM), TPA-S-C2 (10 μM), TPA-S-C4 (10 μM), TPA-S-C6 (10 μM), TPA-S-C8 (10 μM), TPA-S-C10 (10 μM), and TPA-S-C6-NMe3 + (10 μM) upon white light irradiation (Halogen lamp, 25 mW/cm2). Sample mixtures (ABDA + various probes in solution) were then irradiated under white light irradiation (Halogen lamp, 25 mW/cm2) at intervals of 10 s for 300 s. The absorbance spectra changes of various samples were recorded on a UV–vis spectrophotometer. Microbial culture E. coli O157:H7 (Gram negative, ATCC 43894), S. aureus (Gram positive, ATCC 25923), ESBL E. coli (Gram negative, ATCC BAA-198), and C. albicans (Fungi, ATCC 10231) were used for the antimicrobial test. Strains of four kinds of bacteria were grown overnight on an agar plate by the streaking technique to get a single colony. A few isolated colonies from the agar plate were cultured for 3–6 h in 4 mL of broth culture (Luria–Bertani, LB Broth) at 37 °C with a shaking speed of 200 rpm. To monitor the concentration of bacteria, the optical density (OD) at 600 nm was used. Before the following bacterial experiments, the OD 600 nm of the bacterial solution was regulated to 1.0. Photodynamic antimicrobial effect Bacteria were harvest by centrifuging at 5000 rpm and were washed with phosphate-buffered saline (PBS) three times. Then, the bacteria precipitate from 1 mL broth was resuspended with 1 mL of distilled deionized water (DW) to make a stock solution of bacteria. To make the samples for antibacterial determinations, 100 μL of bacterial stock solution were mixed with TPA-S-C6-NMe3 + or MB at various concentrations (0–3 μM) in the 1 mL sample, and they were incubated at 37 °C for 2 h with a shaking speed of 200 rpm. Then, 200 μL of each sample was irradiated with white light (Halogen lamp, 25 mW/cm2) for 10 min. The irradiated samples were diluted 10 times with sterilized DW, and 200 μL of diluted solution was placed on a LB agar plate. Then, plates were incubated overnight at 37 °C. The conventional plate counting method was used to determine the number of viable bacteria. The preparation method of fungal samples was consistent with the methods described above, and the fungal samples were irradiated with white light (Xe lamp, 25 mW/cm2) for 20 min. Cell incubation Human cervical carcinoma cells (HeLa cells) were obtained from the Korean Cell Line Bank (Seoul, South Korea). Cells were cultured in a medium supplemented with 10% fetal bovine serum (Invitrogen) according to the guidelines of the ATCC. The cells were seeded in confocal culture dishes and then incubated for 24 h at 37 °C under a humidified atmosphere containing 5% CO2. MTT assay HeLa cells were used for the cytotoxicity test. A 96-well plate was used for seeding the HeLa cells with minimum essential medium (MEM) culture media. After 24 h, HeLa cells were incubated with TPA-S-C6-NMe3 + (0–3 μM) for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) media (0.5 mg/mL) was added to the 96-well plate with HeLa cells for 4 h. After the addition of 100 μL of dimethyl sulfoxide (DMSO), the plate was read at OD 650 nm with a Spectramax Microwell plate reader. Cell viability (%) was calculated based on the following formula: Cell viability ( % ) = ( OD AIEgen − OD blank ) / ( OD control − OD blank ) × 100 SEM detection Bacteria were harvest by centrifuging at 5000 rpm and were washed with PBS three times. Then, the bacteria precipitate from 1 mL broth was resuspended with 1 mL of a 3 μM TPA-S-C6-NMe3 + solution in sterilized DW, and they were incubated at 37 °C for 2 h with a shaking speed of 200 rpm. Then, 1000 μL of each sample was irradiated with white light (Halogen lamp, 25 mW/cm2) for 10 min. The resultant bacteria suspension was centrifuged, and the supernatant was discarded. Subsequently, samples were fixed in 2% paraformaldehyde at 4 °C overnight. After washing with DW three times, the bacteria were dehydrated in an ethanol series (30%, 50%, 75%, 85%, 95% (once), and 100% (twice) for 10 min each. Then, 10 μL of the dehydrated sample was placed on a piece of silicon wafer. Following drying, platinum was used for coating before SEM inspection. The preparation method of fungal samples was consistent with the methods described above, and the fungal samples were irradiated with white light (Xe lamp, 25 mW/cm2) for 20 min. Confocal microscope detection Bacteria were harvest by centrifuging at 5000 rpm and were washed with PBS three times. Then, the bacteria precipitate from 0.5 mL broth was resuspended with 0.5 mL of a 3 μM TPA-S-C6-NMe3 + solution and 1 μg/mL Hoechst 33342 in sterilized DW, and they were incubated for 30 min. Each sample was placed on a microscope glass slide. The resultant bacteria suspension was centrifuged and the supernatant was discarded. The preparation method of fungal samples was consistent with the methods described above. Zeta potential measurements Bacteria were harvest by centrifuging at 5000 rpm and were washed with PBS three times. Then, the bacteria precipitate from 1 mL broth was resuspended with 1 mL of 1 μM TPA-S-C6-NMe3 + solution in sterilized DW to make a stock solution of bacteria, and they were incubated at 37 °C for 10 min with a shaking speed of 200 rpm. After incubation, the solutions were centrifuged, washed by DW, and used for ζ potential. The preparation method of fungal samples was consistent with the methods described above. Mice model infected with S. aureus BALB/c mice (∼20 g) were anesthetized. Then, a 1.0 × 1.0 cm2 open excision wound was imposed on each mouse via removal of the dorsal flank skin on the spine. Next, 50 μL of S. aureus (108 CFU/mL) was infected over each wound from different groups. For the groups with AIEgen TPA-S-C6-NMe3 + treatment, 50 μL of TPA-S-C6-NMe3 + (3 μM) was then added onto each wound for 20 min. Then, the wound was subjected or not to white light irradiation (Xe lamp, 50 mW/cm2) for 30 min. Please note that the TPA-S-C6-NMe3 + addition and white light irradiation were performed once a day for 3 days and the wound regions were photographed by a camera. On day 6, the infectious tissues of the mouse were separated and homogenized in normal saline and then diluted 1000 times with normal saline to determine the amount of the bacteria in the infectious tissues of mice. Additionally, bacteria solution (20 μL) was sprayed onto a LB agar plate and cultured at 37 °C for 24 h to count the bacterial colonies. Meanwhile, the body weight of mice were also recorded using an analytical balance. Histological examination On day 6, the obtained wound tissues from different groups were first fixed in 4% paraformaldehyde for the histological analysis and were then embedded in paraffin. Next, hematoxylin and eosin (H&E) staining was carried out according to the standard protocols.30 Care and use of animals This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals. The animal protocol was approved by the local research ethics review board of the Animal Ethics Committee of Dalian University of Technology (certificate number/ethics approval no. 2018-043). Female BALB/c mice, about 20 g, were purchased from the specific pathogen free (SPF) Experimental Animal Center of Dalian Medical University. Results and Discussion Spectroscopic characteristics and antimicrobial pathogen evaluation With these compounds in hand, photophysical properties of TPA-S-C0 and TPA-S-C n (n = 0, 2, 4, 6, 8, and 10) were initially studied by UV–vis absorption and fluorescence spectroscopy. As shown in Figure 1a, TPA-S-C0 exhibited an obvious absorption peak around 420 nm. TPA-S-C n with different length alkyl chains showed similar absorption maxima at 536 nm, accompanied by a remarkable absorption shift of 116 nm due to a reduction in the energy level difference between the excited state and the ground state. As alkyl chain length increased, the fluorescence intensities of TPA-S-C n at ∼760 nm gradually increased (Figures 1b and 1c), which was ascribed to the increasing hydrophobicity of the compounds leading to greater aggregation in DW. The triphenylamine (TPA) group of the TPA-S-C n acts as an electron donor (D), and the quinoline cation serves as an electron acceptor (A) to endow the molecules with AIE and NIR emission properties. To verify the AIE features of TPA-S-C n, their spectra were obtained in mixtures of fluorescence intensities increased the of the due to the formation of aggregates, AIE (Figures and and Supporting Information Figures Additionally, the singlet oxygen generation capacity of TPA-S-C n was in DW with white light irradiation mW/cm2). a commonly used 1O2 was used to 1O2 generation has a absorption peak at nm, and 1O2 can its chemical structure ( Supporting Information Figure resulting in of the absorption In to the 1O2 production capacity of the common commercial PSs including Ce6 and were also under the As shown in Figure and Supporting Information Figure the 1O2 production capacity the of TPA-S-C6 TPA-S-C8 TPA-S-C4 TPA-S-C2 TPA-S-C10 Ce6 TPA-S-C0, which that our designed AIEgens efficiently promoted 1O2 generation because of the of a intramolecular effect, which increased the their potential for use in Figure 1 | absorbance spectra of (n = 0, 2, 4, 6, 8, and 10) in Fluorescence spectra and of (10 n = 2, 4, 6, 8, and 10) in DW, = nm. Fluorescence spectra changes of TPA-S-C2 (10 μM) in mixtures of at an of nm. of emission of (10 n = 2, 4, 6, 8, and 10) in mixtures of with of nm. singlet oxygen production capacity of PSs (10 Ce6 and and (10 n = 2, 4, 6, 8, and 10) using as a 1O2 with white light irradiation LB agar plate of S. aureus treatment with (0.5 n = 2, 4, 6, 8, and 10) under or white light irradiation mW/cm2) for 10 min. of E. coli treatment with n = 0, 2, 4, 6, 8, and 10) under or white light irradiation mW/cm2) for 10 min. Download figure Download PowerPoint The photodynamic antibacterial of compounds were The common S. aureus (Gram positive) and E. coli (Gram negative) were first used for As in Figure S. aureus with 0.5 μM TPA-S-C n under white light irradiation mW/cm2) for 10 min exhibited different and a relatively number of bacterial colonies were under incubation with TPA-S-C4 and also that TPA-S-C6 μM) the obvious inhibitory effect on E. coli colonies Additionally, TPA-S-C0 not inhibitory effect on S. aureus and E. coli at a concentration to 3 μM upon white light irradiation ( Supporting Information Figures and Based on the above AIEgen TPA-S-C6 the best antibacterial among TPA-S-C A for the different antibacterial activities among TPA-S-C n is the synergistic effect of alkyl chain length and reactive oxygen production Next, ESBL E. coli, a antibiotic-resistant bacterial was for to the photodynamic antibacterial effect of AIEgens TPA-S-C Unfortunately, all AIE agents were under light irradiation against ESBL E. coli, as depicted in Supporting Information Figure which inspired us to to the molecular structure to endow it with powerful antimicrobial properties. of cationic AIEgens and antimicrobial pathogen evaluation Cationic with groups have been successfully used to to pathogenic the of the evolution of AIEgen Therefore, the optimized TPA-S-C6 was rationally by to a novel to as TPA-S-C6-NMe3 The absorption spectra of TPA-S-C6-NMe3 + in different , and showed obvious which was attributed to the of a Additionally, the aggregation of TPA-S-C6-NMe3 + was also in an undesirable As in Figure the fluorescence of TPA-S-C6-NMe3 + was increased to that AIE characteristics were and SEM the formation of aggregates of cationic TPA-S-C6-NMe3 + in the of aggregates was by the effect of TPA-S-C6-NMe3 + ( Supporting Information Figure The AIEgen TPA-S-C6-NMe3 + with two positive was in an solution and a of at μM Fluorescence spectra were also As in Figure no obvious fluorescence was that the obtained TPA-S-C6-NMe3 + good Figure 2 | absorbance spectra of AIEgen TPA-S-C6-NMe3 + (10 μM) in different and Fluorescence spectra changes of AIEgen TPA-S-C6-NMe3 + upon gradually increasing of analysis of AIEgen TPA-S-C6-NMe3 + in SEM of AIEgen TPA-S-C6-NMe3 + in between the absorbance of TPA-S-C6-NMe3 + and different concentrations μM) in DW. Fluorescence spectra changes of TPA-S-C6-NMe3 + μM) in DW with at nm. Download figure Download PowerPoint Before the photodynamic antimicrobial the 1O2 generation capacity of amphiphilic TPA-S-C6-NMe3 + was As shown in Supporting Information Figure the absorption peak at nm of in the + group upon white light irradiation mW/cm2) with the control group ( Supporting Information Figure and its was that of the commercial groups including ( Supporting Information Figure and + ( Supporting Information Figure of a high 1O2 production capacity ( Supporting Information Figure Meanwhile, the absorption of amphiphilic TPA-S-C6-NMe3 + under white light irradiation for 30 min ( Supporting Information Figure the striking of TPA-S-C6-NMe3 by these we then the photodynamic antimicrobial pathogen efficacy of amphiphilic TPA-S-C6-NMe3 As in Figures there were almost no S. aureus colonies on a LB agar plate addition of TPA-S-C6-NMe3 + at different concentrations 1, and 2 μM) with white light irradiation for 10 min. TPA-S-C6-NMe3 + exhibited of the formation of E. coli colonies under the irradiation (Figures and E. coli colonies on a LB agar plate a significant with TPA-S-C6-NMe3 + Figure even in the of light irradiation, that amphiphilic AIEgen TPA-S-C6-NMe3 + showed an for the of E. coli colonies. A for is the of a between the on the outer member of E. coli and TPA-S-C6-NMe3 + that to severe Additionally, the cytotoxicity of TPA-S-C6-NMe3 + by a standard MTT method that TPA-S-C6-NMe3 + no obvious cytotoxicity ( Supporting Information Figure even at a high concentration of 3 which could be employed for the of E. coli. As amphiphilic TPA-S-C6-NMe3 + powerful PDT against antibiotic-resistant bacterial strain ESBL (Figures which could the of the bacterial colonies were almost by addition of the amphiphilic AIEgen TPA-S-C6-NMe3 + (0.5 Figures and under white light irradiation for 10 min. This is superior to AIE antibacterial an