Host–Guest Interaction Driven Peptide Assembly into Photoresponsive Two-Dimensional Nanosheets with Switchable Antibacterial Activity

Open AccessCCS ChemistryRESEARCH ARTICLE1 Jul 2021Host–Guest Interaction Driven Peptide Assembly into Photoresponsive Two-Dimensional Nanosheets with Switchable Antibacterial Activity Xiaoming Xie, Bo Gao, Zhiyuan Ma, Junqing Liu, Jianfeng Zhang, Jing Liang, Zhijun Chen, Lixin Wu and Wen Li Xiaoming Xie State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Bo Gao State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Zhiyuan Ma State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Junqing Liu School of Life Sciences, Jilin Province Innovation Platform of Straw Comprehensive Utilization Technology, Jilin Agricultural University, Changchun 130118 , Jianfeng Zhang School of Life Sciences, Jilin Province Innovation Platform of Straw Comprehensive Utilization Technology, Jilin Agricultural University, Changchun 130118 , Jing Liang School of Life Sciences, Jilin Province Innovation Platform of Straw Comprehensive Utilization Technology, Jilin Agricultural University, Changchun 130118 , Zhijun Chen State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Lixin Wu State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 and Wen Li *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 https://doi.org/10.31635/ccschem.020.202000312 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Selectively controlling the bioactivity of antimicrobial peptides is not only a fascinating scientific challenge but also a necessity in localized antibacterial therapy. Here, a smart antimicrobial system has been fabricated via host–guest driven dynamic self-assembly between a branched cyclodextrin and cationic linear peptides appended with azobenzene side chains. The self-assembly structure of the host–guest system could be controlled reversibly through the photoresponsive isomerization of azobenzene moieties. Notably, trans-azobenzene side chains of the cationic peptides can interact with the branched cyclodextrin and form microscale sheet-like structures with high surface potentials. The multivalent positive charges covering the surface of the sheet-like structures enable the antibacterial features. However, the UV-triggered cis-isomerization of azobenzene residues weakens the host–guest interactions between azobenzene and the branched cyclodextrin, resulting in the formation of small and inactive nanospheres. Thus, the selective regulation of the antibacterial activity of these peptide assemblies was achieved by delivering the light with spatiotemporal precision. This kind of photoresponsive peptide self-assembly system with switchable bioactivity may provide a new insight into the development of smart supramolecular antibacterial materials. Download figure Download PowerPoint Introduction Bacterial infection remains a major threat to global health and has become aggravated with the unceasing proliferation of antimicrobial resistance.1–3 The emergence of the antibiotic crisis demands the development of new approaches and antimicrobial agents, which are not affected by the conventional mechanisms of antibiotic resistance in bacteria.4–6 In this context, antimicrobial peptides (AMPs), serving as endogenous host defense molecules in many living organisms, are highly promising candidates.7–12 Unlike conventional antibiotics that exert the antibacterial function relying on a receptor-specific target manner, most AMPs carrying positive charges can attach to negatively charged bacteria cell membranes via nonspecific electrostatic interactions.13–15 The AMP segments binding on the surface of bacteria will accumulate and subsequently enter the bacterial cell to destroy the membrane, thereby causing the leakage of cytoplasmic components as well as the apoptosis of bacteria.16,17 Because AMPs have nonspecific action mechanisms, much research has commenced on the design and synthesis of various cationic short peptides for antibacterial treatment.18–21 However, even with these advances, the poor activity and stability of the short peptides under physiological conditions greatly plague their application. Peptide assembly has a significant role in the creation of bioactive materials with exquisite levels of efficiency.22–31 Recent studies report that self-assembling AMPs with multivalent nanostructures are highly effective in killing bacteria.32–36 The highly concentrated cationic groups covering the surface of the AMP nanostructures can enhance their binding affinity to bacteria, and lower the dose of AMPs required to treat bacteria.37 Additionally, molecular stacking within the nanostructures largely limits enzyme accessibility, thus improving the stability of AMPs against proteolytic hydrolysis.38 More importantly, the dynamic assembly and disassembly features of the AMPs provide great opportunity to engineer their antimicrobial efficacy on demand.39–41 For example, Chen et al.39 reported a guest-triggered AMP assembly system with switchable antibacterial activity. Yang et al.40 harnessed the overexpressed phosphatase in Escherichia coli to catalyze the assembly of short peptide or antibiotic-peptide conjugates,42 activating their biological functions within the bacteria cell. Chen et al.41 developed a pH-responsive AMP nanomaterial, which could be activated only in the acidic microenvironment of the bacterial infection area. These vivid examples further impel us to develop smart antibacterial agents with controllable bioactivities with spatiotemporal precision, which is crucial for implementing localized antibacterial therapy.43,44 Light is a noninvasive and bio-orthogonal trigger, and can be delivered with highly spatial and temporal selectivity.45–47 A pioneering study on the spatiotemporal control of antibacterial activity has been reported by Velema et al.48 and Wegener et al.49 via coupling conventional antibiotics with a photoactive azobenzene moiety. Recently, Babii et al. reported smart AMPs by introducing a diarylethene unit into a cyclic peptide backbone. The photoswitchable backbone conformation of the cyclic peptidomimetics enables selective control of antibacterial activity at the molecular level.50 However, the development of cyclic AMPs with photoresponsive behavior remains a great challenge and is difficult to generalize due to the rigorous design requirement. It also suffers from inherent problems associated with complicated synthesis and poor yield. As the self-assembling AMPs are based on dynamic noncovalent interactions, it is very logical to develop a simple but universal strategy to generate smart AMPs based on relatively simple linear peptides by taking advantage of their photoswitchable self-assembly at the supramolecular level. To address this issue, a series of azobenzene-containing linear peptides (P1–P4, Figure 1a) and a trigeminal β-cyclodextrin (tri-β-CD, Figure 1a) were designed and synthesized. It is expected that the azobenzene side chains of these peptides will recognize the tri-β-CD, forming cross-linking assemblies via host–guest interactions. The photoresponsive isomerization of azobenzene moieties allows us to dynamically regulate the assembly and disassembly between the synthesized peptides and tri-β-CD. The lysine side chains are introduced into the molecular design to provide nonspecific binding sites to bacteria, and the hydrophobic residues (such as valine, isoleucine, and alanine) facilitate peptides insertion into the cell membrane of the bacteria.15,17 Herein, we report the host–guest interaction driven self-assembly of the cationic peptides and tri-β-CD, and their enhanced antibacterial potency. We further demonstrate how the antibacterial activity of the peptides and tri-β-CD complexes could be controlled with spatiotemporal precision via photoswitchable self-assembly. Figure 1 | (a) Structures of peptides P1–P5 and the tri-β-CD. (b) The schematic drawing of the photoresponsive assembly between P1 and tri-β-CD. Download figure Download PowerPoint Experimental Methods Preparation of peptides/tri-β-CD complexes The peptides and tri-β-CD solutions were first prepared by dissolving the lyophilized peptides and tri-β-CD powders in deionized water, respectively. Then, the tri-β-CD solution was added dropwise into the stock solution of peptides, and the resulting solution was sonicated at 25 °C for 3 min. The molar ratio of peptides to tri-β-CD was maintained at 3∶2 and the solution was maintained at approximately 7.0 pH. The self-assembly peptides/tri-β-CD complexes were obtained after aging the mixed solution for ca. 3 h at 25 °C. The photoresponsive self-assembly was performed by exposing the aqueous solution of peptides/tri-β-CD to UV (365 nm) or visible light (470 nm) for 10 min. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was captured on an Autoflex speed TOF/TOF (Brucker) operating in positive mode within a mass range from 700 to 5000 Da. Proton nuclear magnetic resonance Proton nuclear magnetic resonance (1H NMR) spectra were collected on a Bruker AVANCE 500 MHz spectrometer by dissolving P1, tri-β-CD, and P1/tri-β-CD in D2O at 25 °C. Two-dimensional (2D) 1H Nuclear Overhauser Effect Spectroscopy (NOESY) spectra were recorded on a Bruker AVANCEIII 600 MHz spectrometer, and the optimized mixing time was set to 200 ms before the acquisition of the free induction decay. The trans-P1/tri-β-CD (the molar ratio of P1 to tri-β-CD is 3∶2) solution was prepared freshly in H2O/D2O (v/v = 9∶1) and kept at room temperature for 2 h before NOESY testing. The cis-P1/tri-β-CD sample was prepared by exposing the trans-P1/tri-β-CD solution to UV light (365 nm) for 30 min before NOESY testing. The concentration of P1 in all the solution sample was kept at 8 mg mL−1. Dynamic light scattering Dynamic light scattering (DLS) experiments were recorded at 25 °C on a Malvern Zetasizer Nano ZS (Malvern Instruments, UK) using a detection angle of 173° and a 3 mW He–Ne laser operating at λ = 633 nm. The temperature equilibration time was set to 120 s in all cases, and the measurements were repeated at least three times. Circular dichroism Circular dichroism (CD) spectra were recorded on a JASCO model J-810 spectropolarimeter (25 °C, Xe lamp) under a constant flow of nitrogen gas during operation. The solution samples were loaded into a rectangular quartz cell with a 0.1 cm path length, and the CD spectra wavelength range was 260–190 nm with a step of 0.5 nm. The analyses were repeated five times and averaged. The JASCO software was used for background subtraction. Thioflavin T binding study The thioflavin T (ThT) titration was measured using the following procedure. A ThT solution was added into the aqueous solution of P1, and the final concentration of ThT was maintained at 10 μM. The resultant P1 solution (pH ∼7) was incubated at room temperature for 5 h. The fluorescence spectra of individual ThT (10 μM) and the P1/ThT solution samples were recorded on a 5301 PC spectrophotometer (Shimadzu, Tokyo, Japan), respectively, with an excitation wavelength of 420 nm. Zeta potential The zeta potentials of the P1/tri-β-CD solution samples were measured using the Zetasizer NanoZS instrument (Malvern Panalytical) at 25 °C. UV–visible spectra The UV–visible (UV–vis) spectra were performed on a Varian Cary 50 UV–vis spectrophotometer. The wavelength is in the range of 200–600 nm with a step of 1 nm. Transmission electron microscopy The transmission electron microscopy (TEM) images were acquired on a JEOL-2010 electron microscope (200 kV). The peptides/tri-β-CD solution samples were casted onto a carbon-coated copper grid and then dried completely in air. The individual peptide samples and the peptides/tri-β-CD specimens irradiated by UV light (365 nm) were stained, respectively, with 0.1 wt % uranyl acetate aqueous solution for 3 min, the excess amount of the uranyl acetate solution was removed by filter paper. The trans-peptides/tri-β-CD specimens without staining were used directly for TEM measurements. E. coli cells with and without peptides/tri-β-CD were washed with phosphate-buffered saline (PBS) at least three times. The centrifuged E. coli samples were redissolved in deionized water and casted on a carbon-coated copper grid for TEM measurements. Cryogenic TEM Cryogenic TEM (Cryo-TEM) was performed on a JEOL-JEM 2100 TEM instrument (approximately 90 K, 120 kV) equipped with a SC 1000 charge-coupled device (CCD) camera (Gatan, Inc., USA). A liquid droplet of P1/tri-β-CD (3 μL) was transferred to an ultrathin copper grid after hydrophilic treatment under controlled temperature and humidity (97–99%) to prevent evaporation of sample solution. Then, the superfluous liquid droplets were removed with filter paper, and the thin aqueous films were rapidly vitrified by plunging them into liquid ethane and cooled to approximately 90 K by liquid nitrogen. The excess amount of ethane was removed using blotting paper after the sample solution was frozen. Finally, the grid was inserted into a Gatan 626 cryo holder using a cryotransfer device for cryo-TEM measurements. Atomic force microscopy Atomic force microscopy (AFM) measurements were recorded on a Bruker Dimension 3100 instrument (Karlsruhe, Germany) using a tapping mode in air (25 °C). The AFM samples were prepared by casting the P1/tri-β-CD solution on the fresh surface of a mica wafer. After settling for 3 min, the excess amount of solution was removed by filter paper, and the air-dried samples were utilized for AFM tests. Laser scanning confocal microscopy Laser scanning confocal microscopy (LSCM) measurements were carried out using a FV1000 confocal microscopy. The P1/tri-β-CD solution (60 μM) was incubated with Rhodamine B solution (10 μM), a fluorescence probe exhibiting an increase of emission when it is adsorbed on the surface of P1/tri-β-CD nanosheets. The resultant solution was casted on glass for LSCM measurements with an excitation wavelength of 515 nm. In the case of the dead assay for cell viability, the E. coli cells were treated with propidium iodide (PI; 0.05 mg mL−1) and fluorescein diacetate (FDA; 0.02 mg mL−1) for 30 min, and then washed with PBS buffer at least three times before the test analysis. The obtained solution of double staining E. coli was casted on glass for LSCM observation with an excitation wavelength of 488 and 515 nm, respectively. The emission wavelengths were fixed at 520–550 nm (for FDA) and 600–650 nm (for PI), respectively. Scanning electron microscopy Scanning electron microscopy (SEM) images were obtained on a JEOL FESEM 6700F electron microscope (15 kV). E. coli cells treated with and without P1/tri-β-CD were washed with PBS buffer at least three times. The centrifuged E. coli was redissolved in deionized water and casted on a clean silica wafer adhered onto an aluminum sample holder and dried in the air, then sputter coated with platinum. Antibacterial assays Fresh Luria–Bertani (LB) liquid medium was obtained by mixing 2.5 g of peptone, 1.25 g of yeast extract, and 2.5 g of sodium chloride in 245 mL of deionized water and sterilized at 121 °C for 20 min. The LB agar plate was obtained by adding another 3.75 g agar powder into the LB liquid medium and then sterilizing at 121 °C for 20 min. Bacterial strains (Gram-negative: E. coli and Pseudomonas aeruginosa, Gram-positive: Staphylococcus aureusand Bacillus subtilis) were suspended in 25 mL of LB liquid medium (containing 25 μL of ampicillin), respectively. The obtained samples were cultivated in a constant temperature shaker (37 °C, 160 rpm) for 12–14 h to reach the stationary growth phase. The resulting bacterial broth was diluted to ∼0.035 of the OD600 (BioPhotometer plus instrument), then 2.0 mL of bacterial cell suspensions were added into four glass tubes containing deionized water (as the blank test), peptides, tri-β-CD, and peptides/tri-β-CD, respectively. The solution volume of each tube was kept at 2.5 mL, the concentrations of peptides were maintained at 5–60 μmol L−1, and the concentration of tri-β-CD was kept at 3.3–40.0 μmol L−1. The antibacterial activity of the samples was assessed through monitoring the OD600 values of the bacterial cell suspensions at indicated time points by the BioPhotometer plus instrument with a 10 s shaking step before each measurement. The antibacterial activity of the E. coli samples treated with P1/tri-β-CD was also evaluated by counting the colony-forming unit (CFU). The treated E. coli suspensions were serially diluted 1 × 10n fold with PBS buffer solution, then a 100 μL portion of the diluted solution was spread on the solid LB (supplemented with 1 μL mL−1 ampicillin) agar plate. After an incubation for 20 h at 37 °C, the final colonies of E. coli were counted. The tests were repeated three times and averaged. In the case of the photoresponsive antimicrobial test with temporal selectivity, the LB liquid medium containing trans-P1/tri-β-CD samples was first irradiated at 365 nm for 10 min in the dark prior to adding the E. coli cell suspension (OD600 ≈ 0.035), then the samples were irradiated by visible light (470 nm, 10 min) with different time intervals (0, 84, and 144 min). All samples after irradiation were still placed in an incubator shaker (160 rpm, 37 °C) to cultivate. In the case of the photoresponsive antimicrobial test with spatial selectivity, the LB liquid medium containing E. coli cells at the stationary growth stage was diluted to OD600 ≈ 0.035, and a 20 μL portion of the diluted E. coli was coated uniformly on the surface of LB agar plate containing trans-P1/tri-β-CD. After waiting for 15 min, the agar plate was kept in the dark and irradiated with UV light for 10 min (365 nm, 6 W). Next, the agar plate was illuminated with visible light (470 nm) through a mask placed on top of the plate. Afterwards, the plate was incubated in a constant temperature shaker (37 °C) overnight to allow for bacterial growth. For the antimicrobial test with spatiotemporal resolution, diluted E. coli culture droplets (OD600 ≈ 0.035) were coated uniformly on the surface of a LB agar plate containing cis-P1/tri-β-CD. The LB agar plate was kept in the dark and covered with a rotatable mask. The sample was incubated at 37 °C and irradiated (470 nm, 10 min) selectively at different areas by rotating the mask with different time intervals. After incubation in the dark for 24 h, patterned sectors of E. coli were observed. Cytotoxicity assay L-929 cells (mouse fibroblasts) were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and 1% penicillin/streptomycin. The assays were carried out in sterile 96-well flat-bottomed polystyrene microtiter plates. Plates containing 100 μL of cell suspension in each well (5000 to 10,000 cells/well) were preincubated for 24 h at 37 °C in a humidified environment with 5% CO2. The samples to be tested were two-fold diluted, and a 10 μL portion of the tested compounds (P1, tri-β-CD, and P1/tri-β-CD) was added to test plates in triplicate to get a final concentration of 60 μM (79.8 μg mL−1). The plates containing the tested compounds were incubated for 24 h. Subsequently, the plates were further incubated with 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,3-dihydro-1H-tetrazol-3-ium bromide (MTT) solution (2.5 mg mL−1) at 37 °C for 4 h. The top medium was then removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The absorbance of the solution was measured with an enzyme standard instrument at 570 nm using a multiwall plate reader (AccuSkan, MultiSkan FC, Thermo Fisher Scientific). For fluorescence microscope examination, L-929 cells were inoculated in a 96-well flat-bottomed polystyrene microtiter plate with the number of cells at approximately 104, 5 × 103, and 103, respectively. The liquid medium was changed, replaced with fresh medium after inoculation for 24 h, and then P1/tri-β-CD was added. The live cell assay was monitored with different incubation time (1, 3, and 7 days, respectively). Afterwards, the cells were washed using PBS solution two to three times to remove the active esterase from the medium. Dye-working fluid (100 μL) containing 2 M calcein AM and 8 M PI of PBS was added to each hole, then incubated at 37 °C for 30 min. Finally, the dye-working fluid was extracted and visualized under a FV1000 confocal microscopy. The excitation wavelengths were fixed at 490 and 545 nm, respectively. Results and Discussion Host–guest interaction and self-assembly The linear peptide P1, which of an of hydrophilic and hydrophobic residues was synthesized by a standard peptides Figure 1a) with various residues were also prepared to the of on the antibacterial activity. Peptide without an azobenzene was synthesized for The of all the peptides was by liquid Figure and Figure The tri-β-CD was synthesized the reported and was by 1H Figure and Figure of the P1 aqueous solution (pH a of nm Figure a solution of P1 was onto a copper grid and then negatively with uranyl structures with a of nm were by The CD with a at nm Figure that P1 a conformation in aqueous solution (60 This structure of P1 was by ThT titration Figure We that the conformation of P1 from the electrostatic of the lysine residues The conformation of cationic P1 the of tri-β-CD to an aqueous solution of P1 even with an molar ratio of P1 to tri-β-CD of as from the CD Figure However, of the P1/tri-β-CD sample (60 μM) a of Figure which is much that of individual TEM of the P1/tri-β-CD sample further the formation of assemblies at the The AFM of the P1/tri-β-CD sample the as an ultrathin with a of nm. To the of water evaporation on the we performed the study by cryo-TEM and As in and the cryo-TEM and the LSCM that the in the aqueous solution on a solid surface during the This formation of was not to P1/tri-β-CD azobenzene-containing peptides and also ultrathin when mixed with tri-β-CD Figure In or were in the case of due to the of azobenzene residues Figure The that the are by the host–guest between tri-β-CD and the azobenzene residues of the Figure 2 | (a) TEM of (b) TEM of AFM and of P1/tri-β-CD Download figure Download PowerPoint This was by 1H The of the of tri-β-CD and P1 in D2O are respectively, in and P1 molecules and at which were to the of trans-azobenzene In the case of P1/tri-β-CD the azobenzene residues due to the resulting from the formation of via the host–guest interactions between azobenzene moieties and tri-β-CD. To the host–guest of the P1/tri-β-CD 1H NOESY were As in Figure the in the A are to the between the of the azobenzene of P1 and the or in the of and the in the B to the between the of the azobenzene of P1 and the in the of These demonstrate that the trans-azobenzene moieties of P1 are in the hydrophobic of tri-β-CD via host–guest that the P1/tri-β-CD aqueous sample a potential of thus that the surface of the was covered by highly concentrated and lysine residues of the conformation of P1, the molecular nm) of P1 between the unit and the hydrophilic at the of the nm of the nm of the and the high zeta we a assembly As in Figure the tri-β-CD segments as to the P1 molecules via host–guest interactions, the supramolecular However, the lysine groups with highly concentration on the surface of the assemblies have a to molecular due to the of electrostatic The of these noncovalent interactions to the formation of with lysine groups on the top and surface in aqueous solution. The assembly was by the following series of when P1 was mixed with under the small nm, Figure with zeta potential Figure were that the β-cyclodextrin is to the cross-linking assembly and supramolecular the formation of the of P1/tri-β-CD in aqueous solution is As in Figure structures with of nm were when the P1/tri-β-CD solution sample was prepared at Notably, this solution sample was and has to into with structures at the Figure However, with the to the can into the solution within 5 min, and with Figure were within 1 h. aging time 3 the and were Figure This between and structures indicated that the electrostatic the lysine groups is to the formation of