Article Cite This: Chem. Mater. XXXX, XXX, XXX−XXX
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Biodegradable Synthetic Antimicrobial with Aggregation-Induced Emissive Luminogens for Temporal Antibacterial Activity and Facile Bacteria Detection Shuai Chen,†,# Qixian Chen,‡,# Qiaoying Li,† Jinxia An,† Peng Sun,‡ Jianbiao Ma,† and Hui Gao*,† †
School of Chemistry and Chemical Engineering, Tianjin Key Laboratory of Organic Solar Cells and Photochemical Conversion, Tianjin University of Technology, Tianjin 300384, P. R. China ‡ School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, P. R. China S Supporting Information *
ABSTRACT: Aiming to address the antimicrobial resistance and environmental burdens because of the widespread indulgence in biocides, a synthetic formula of biocides with controllable antibacterial activities and biodegradable characters are encouraged for the emergence of a novel spectrum of green biocides. In this respect, a polymeric antimicrobial catiomer, with the polymeric backbone sequentially attached with the functional cationic moieties and lipophilic moieties is synthesized. The striking antibacterial potency is achieved for treatment of both amoxicillin (AMO)-resistant Gram-positive S. aureus and Gram-negative E. coli. Moreover, the anionic aggregation-induced emission (AIE) compound is strategically introduced into the aforementioned polymeric antibacterial system, consequently employed as a facile reporter for the utility of bacteria detection based on the electrostatic reaction of anionic species in the bacterial membrane with the cationic polymeric antibacterials to liberate the anionic AIE compound. Ultimately, the proposed system is determined to afford appreciable temporal antibacterial potency by virtue of the readily available cleavage of the ester linkage in the presence of lipase, thereby eliminating the possibility of the antimicrobial resistance and environmental burdens. Hence, the proposed system provided a bottom-up approach for construction of an intriguing green antibacterial molecule.
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INTRODUCTION The discovery of penicillin has led to the excitement of pharmaceutical chemists with respect to its intriguing promise in eradication of bacteria-related diseases, which spurred myriad of spectra of antimicrobial drugs for the utilities of prevention and treatment of bacterial infections.1−5 The tempting medical potency has led to constant bombardment of antimicrobial drugs in the medical uses. Nevertheless, overuse of antibiotics and inappropriate antibiotic treatment have caused the emergence of antibiotic-resistant bacteria, sometimes referred to as “superbugs”, for which the previously effective antibacterial treatments are losing their effectiveness, consequently imposing tremendous therapeutic challenges.6,7 For instance, approximately 500 000 new cases of multidrugresistant tuberculosis are estimated to occur worldwide annually.8 Moreover, the poor degradability of antibacterials has resulted in widespread environmental, agricultural, and biological chain problems and eventually may pose marked threats to human health.9 In an attempt to resolve the antimicrobial resistance and the environmental concerns, synthetic antibacterials with temporal antibacterial activities and readily biodegradable properties are emphasized for pursuit of green biocides. To this end, we © XXXX American Chemical Society
attempted to tailor a polymeric antimicrobial formula, where the polymeric backbone was attached with the functional segments, including: (i) cationic amino segments enable readily available interactions with the cell membrane of bacteria and induce the membrane structural destabilization; (ii) lipophilic alkane segments afford appreciable assistance in the association potency of the polymeric antimicrobials to the cell membrane of bacteria and distortion potency to the membrane structure of bacteria. Aside from strategic design in promoting antibacterial potency, ester linkages were introduced between the functional segments with aims of possessing antibacterial activity in a temporal manner given that ester bond is subjected to appreciable hydrolysis, particularly susceptible to esterase (e.g., lipases) in the biological milieu. Consequently, the degradable character is speculated to be favorable in minimizing the possibilities of the induced antimicrobial resistance, environmental burdens, and the threat of antibacterial accumulation to human health along the biological chain. Received: January 18, 2018 Revised: February 8, 2018 Published: February 14, 2018 A
DOI: 10.1021/acs.chemmater.8b00251 Chem. Mater. XXXX, XXX, XXX−XXX
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Chemistry of Materials
Scheme 1. Schematic Illustration of Tailoring the Well-Defined Polymeric Q-PGEDA-OP/TPE Antimicrobial System for the Bacterial Detection and the Antibacterial Activity of the Q-PGEDA-OP prior to and after Hydrolysisa
a
The nanoparticles fabricated from a polymeric antimicrobial catiomer with the aggregation-induced emission luminogens (AIEgens) through electrostatic interactions. Upon exposure to bacteria, the competitive electrostatic interactions could induce the release of AIEgens, thereby affording facile bacteria detection function. Moreover, the cleavage of ester linkage would produce the anionic carboxylic groups, which is assumed to decrease the association to the bacterial cytoplasmic membrane based on electrostatic repulsion, thereby the antimicrobial polymer degraded into inactive one. Preparation of Q-PGEDA-OP/TPE Nanoparticles. The synthesis procedure of TPE was described in Supporting Information. To obtain the nanoparticles at varying TPE/Q-PGEDA-OP mass ratios (1:20, 1:10, 1:5, 1:2.5, and 1:1), the PBS solutions (pH = 7.4) with TPE at various concentrations (1, 2, 4, 8, or 20 mg mL−1) were prepared, followed by addition of 20 mg of Q-PGEDA-OP, and stirred for 30 min. Bacterial Culture. AMOr S. aureus (Gram-positive) and AMOr E. coli (Gram-negative) were employed as the representative bacteria for test the antibacterial activities of the constructed systems. In brief, the bacteria were cultured into liquid Luria−Bertani (LB) medium (5 mL) on a shaking incubator (37 °C, 170 rpm) overnight. To create resistant variants, the cultures were passaged 30 times in the presence of rising concentrations of AMO based on the growth conditions of the variants. Herein, AMOr S. aureus or AMOr E. coli were referred to the corresponding resistant variants of the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli, respectively. The concentration of the bacteria was quantified on the basis of the measurement of optical density (OD) at λ = 600 nm (note that OD600 of 2.0 was determined to have a concentration of 109 colony forming units per milliliter (CFU mL−1) based on calibration). Determination of Minimum Inhibitory Concentration (MIC). Aiming to determine the minimum inhibitory concentration (MIC), a stock solution of either AMOr S. aureus (Gram-positive) and AMOr E. coli (Gram-negative) were prepared. Briefly, AMOr S. aureus and AMOr E. coli were cultured in 5 mL of liquid LB (supplemented with 50 μg mL−1 AMO) broth at 37 °C on a constant temperature shaker at 170 rpm. After incubation overnight, the concentration of bacterial suspension was diluted with pure nutrient broth to ∼105 CFU mL−1 and measured with a UV-3900 (Shimadzu Co., Japan). A sample of nanoparticles was dissolved in deionized water and diluted from 2000 to 2 μg mL−1 by a series of 2-fold dilutions using nutrient broth. The diluted bacterial solution (100 μL) and the samples (1 mL) with varying concentration were incubated at 37 °C in glass culture tubes, respectively. After incubation for 16 h, the viability of bacteria was determined by measuring OD600 on a Shimadzu UV-3390 instrument. In comparison to a positive control, the minimal inhibitory concentration (MIC) was defined as the lowest polymer concentration that inhibited more than 90% of germ growth.
Moreover, an anionic aggregation induced emission (AIE) component of tetraphenylethene (TPE) derivative was strategically introduced into the aforementioned polymeric antibacterial system, which was utilized as a facile reporter for bacteria detection by virtue of its distinctive AIE property, characterized to afford limited fluorescence emission in an unperturbed status but subjected to pronounced amplification in fluorescence emission when confined in a restricted status. The bacteria detection was envisioned to be as a consequence of AIE status transformation from the restricted complexion with cationic polymeric antibacterials to the liberated molecule status through the exchange reaction of anionic species in the bacterial membrane for competing interaction of anionic AIE component with cationic polymeric antibacterials. Hence, the proposed system with biodegradation nature, temporal antibacterial activity, and facile bacteria detection should be emphasized in development of the next generation of antibacterial drugs and could be further developed to prompt the conceptual green pharmaceutical industry (Scheme 1).
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EXPERIMENTAL SECTION
Preparation of Q-PGEDA-OP. Poly(glycidylmethacrylate) (PGMA) and ethylenediamine-modified PGMA (PGEDA) were synthesized according to a previous report,10 and briefly described in the Supporting Information. Then, PGEDA (100 mg, equivalent to 0.75 mmol NH) was dissolved in 20 mL of H2O/DMSO (1:1) mixture, and then n-octyl acrylate (220 mg, Mw = 184.28, 1.2 mmol) was added dropwise to the solution. After stirring at room temperature for 48 h, the solution was warmed to 45 °C for another 24 h. Consequently, iodomethane (338 mg, Mw = 141.94, 2.4 mmol) was then added to the mixture and stirred in the dark overnight. The final product was first purified by precipitation in diethyl ether twice. Then the crude product was dissolved in 3 mL of distilled water and dialyzed against sodium chloride solution and distilled water for 48 h, respectively, and lyophilized to obtain the octyl-propionate-modified PGEDA quaternary ammonium salt (Q-PGEDA-OP) as white powder (yields: 85%). B
DOI: 10.1021/acs.chemmater.8b00251 Chem. Mater. XXXX, XXX, XXX−XXX
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Chemistry of Materials Scheme 2. Synthetic Scheme for Preparation of the Lipophilic Catiomer of Q-PGEDA-OP
Enzymatic Hydrolyzation of the Polymer and MIC Determination. The hydrolysis of the ester group of the Q-PGEDA-OP was investigated using lipase. Briefly, 20 mg of Q-PGEDA-OP was dissolved in 1 mL of phosphate buffered D2O (0.2 M, pH = 7.0). Then, 2 mg of the lipase was added to the solution at 37 °C. This solution was periodically explored with 1H NMR. As a control, a sample without the lipase, containing only 1 mL of buffer and QPGEDA-OP was prepared. The product after hydrolysis with carboxyl end-groups was shorted as Q-PGEDA-COOH. The MIC determination of these enzymatic hydrolysis samples was performed analogous to the previously described one. Fluorescent Spectra Measurements. The bacteria were cultured in LB media at 37 °C on a constant temperature shaker at 170 rpm overnight. Then the bacteria were centrifuged at 8000 rpm for 3 min and washed with PBS three times. The bacteria were resuspended in PBS and diluted to various concentrations. A solution in PBS (pH = 7.4) of Q-PGEDA-TPE (0.5 mL) were mixed with bacterial suspensions (0.5 mL) of different concentrations (106 to 109 CFU mL−1). The samples were incubated at room temperature for 5 min before the fluorescent spectra were measured. Scanning Electron Microscopic (SEM). To gain insight into the impact of Q-PGEDA-OP hydrolysis on its antibacterial activity to the bacteria (AMOr S. aureus and AMOr E. coli), SEM characterization was employed in this study. Herein, the resistant bacteria were cultured at 37 °C until a concentration of 109 CFU mL−1 was reached. Then, the bacteria were harvested by centrifugation and resuspended in PBS (pH = 7.4). Then the bacteria suspension alone or treated with Q-PGEDATPE or Q-PGEDA-COOH were shaken at 170 rpm at 37 °C for 6 h, followed by being centrifuged at 8000 rpm for 3 min and washed with PBS three times. The samples were solidified with 2.5% glutaraldehyde for 4 h at room temperature. Next, the samples were further washed twice with PBS and dehydrated by addition of ethanol in a graded series (30%, 50%, 70%, 80%, 95%, 100% for 30 min) and then replaced by isopentyl acetate. At last, the samples were coated onto a silicon slice and dried before being observed by SEM. LB-Agar Plates. AMOr S. aureus and AMOr E. coli were seeded into Luria−Bertani (LB) broth for overnight incubation on a shaking incubator (170 rpm) at 37 °C. The bacteria were subjected to centrifugation (8000 rpm for 3 min) and suspended in PBS (1 mL). The bacterial suspensions were serially diluted with PBS to a concentration of 1 × 107 CFU mL−1. Furthermore, the bacteria suspension alone or treated with AMO (200 and 500 μg mL−1 for AMOr S. aureus and AMOr E. coli, respectively) or Q-PGEDA-TPE (at concentration of its MIC) were shaken at 37 °C and 170 rpm for 3 h, followed by being diluted with an appropriate dilution factor. Diluted bacterial suspensions (100 μL) were then plated onto the solid LB agar plates. The colonies formed after 16−18 h incubation at 37 °C
were counted, and the bacterial viability rates were determined. The CFU ratio was calculated according to the following equation: CFU ratio = C/C0 × 100%. C and C0 are the CFU of the experimental group treated with drugs or the control group without any treatments, respectively. The results were presented as a mean and standard deviation obtained from three parallel groups. Fluorescence Microscopic Observation. Around 109 CFU mL−1 of bacteria were harvested and washed with PBS three times by centrifugation (8000 rpm for 5 min) and resuspended in 1 mL of PBS. Then the bacteria alone or treated with of Q-PGEDA-OP were shaken at 170 rpm at 37 °C for 30 min, followed by being stained with 100 μL of fluorescent dye (acridine orange (AO, 1 mg mL−1) and ethidium bromide (EB, 1 mg mL−1) for 30 min. After staining, the bacterial cells were rinsed with PBS three times and imaged via inverted fluorescence microscope (F-4500, Hitachi, Japan). Statistics Analysis. Significant differences in bacteria viability between any two groups were evaluated using Student’s t test.
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RESULTS AND DISCUSSION Characterizations of Synthetic Products. The functional components of anionic carboxyl-functionalized TPE derivative was synthesized according to Scheme S1. The detailed synthetic procedures were carried out according to the previous literature.11 The resulting product of TPE was characterized by 1H NMR measurement, wherein the area integration of the peaks from the obtained 1H NMR of TPE was determined to quantitatively coincide with the protons of the resulting chemical structure of TPE, affirming the successful preparation of TPE (Figure S1). Furthermore, the cationic lipophilic octylpropionate-modified PGEDA quaternary ammonium salt (QPGEDA-OP) was synthesized according to Scheme 2, the precursor PGMA was sequentially attached with the functional moieties of cationic ethylene diamine (EDA) and lipophilic alkane derivatives at its pedants. Note that the polymerization degree of the backbone of PGMA was determined to be approximate 85 units [Mn: 12 kDa; poly dispersity index (PDI): 1.32]. The yielding products were characterized by 1H NMR (Figures S2, S3) and FT-IR measurements (Figure S5). The peaks from the obtained 1H NMR spectrum of Q-PGEDAOP could be quantitatively assigned to the protons of the chemical structure of Q-PGEDA-OP, thereby approving the successful preparation of Q-PGEDA-OP. Moreover, the FT-IR was also conducted for the samples of PGMA, PGEDA, and QPFEDA-OP. The disappearance of absorbance at 991 cm−1 in C
DOI: 10.1021/acs.chemmater.8b00251 Chem. Mater. XXXX, XXX, XXX−XXX
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Chemistry of Materials FT-IR spectrum of PGEDA approved the successful aminolysis of the epoxide group.12 Meanwhile, the emergence of a broad and strong peak at 3400 cm−1 attributed to the presence of the hydrogen bond stretching vibration of hydroxyl and amino functionalities, which gave further verification of the conjugation of cationic EDA segments.13 The emergence of a strong absorbance at 3000−2800 cm−1 in the FT-IR spectrum of Q-PGEDA-OP approved the successful conjugation of lipophilic alkane segments, and the disappearance of N−H stretching vibration band at 1560 cm−1 confirmed the occurrence of quaternization reaction. Additionally, octylamidate-modified PGEDA quaternary ammonium salt (QPGEDA-OA), as a nondegradable control, was synthesized according to Scheme S2, whose 1H NMR characterization (Figure S4) verified its nondegradable nature. Besides, the emergence of amide I and II bands at 1650 and 1540 cm−1 in FT-IR spectrum of Q-PGEDA-OA approved the successful conjugation of nonanoic acid groups (Figure S5). Antibacterial Mechanism Analysis. The synthesized lipophilic Q-PGEDA-OP catiomer together with AIE TPE derivative was used for test its potential antibacterial activities. Pertaining to the structure of bacteria, the main constitution of the cell walls is peptidoglycan, which exhibits net negative charges on the membrane surface.14 The antibacterial activity of the constructed microbial system was assumed to derive from the electrostatic reaction of the constructed lipophilic QPGEDA-OP catiomer (together with TPE derivative) with the anionic cell membrane of the affected bacteria.15 The initial electrostatic interaction of Q-PGEDA-OP and anionic species in the cell membrane of bacteria was conjectured to not only interfere the functions of the affected bacteria but also induce bacteria structural destabilization, consequently accounting for the antibacterial efficacy. Design and Characterizations of Nanoparticles. Herein, the additional luminogens of TPE derivative could serve as a useful functional component to boost the antibacterial activities and detect the existence of bacteria.16,17 Notably, TPE has recently been developed as a novel fluorophore characterized to present distinctive aggregation induced emission (AIE) behavior, whose fluorescence emissive intensity was subjected to marked amplification when its motion was restricted.18−21 In the proposed Q-PGEDA-OP/TPE antibacterial system, anionic TPE was believed to electrostatically associate with cationic QPGEDA-OP, thereby eliciting enhanced fluorescence emission of TPE. The association between Q-PGEDA-OP and TPE was confirmed on the basis of a series of experiments including dynamic light scattering (DLS) and scanning electron microscope (SEM). In brief, as observed in SEM (Figure 1), the QPGEDA-OP nanoparticles prior to TPE conjugation exhibited a uniform size distribution (100 ± 10 nm). Following the complexation with TPE, the resulting formation exhibited marked shrinkage to be 80 ± 6 nm in diameter, which is attributed to the electrostatic interactions between Q-PGEDAOP and TPE.16,22,23 DLS measurement for the Q-PGEDA-OP solution supplemented with varied concentration of TPE observed progressive rise in the scattering intensity (Figure S6), which further confirmed complexation of cationic lipophilic Q-PGEDA-OP and anionic TPE derivatives. Aiming to demonstrate the AIE behavior of the incorporated TPE derivatives, the fluorescence intensity of TPE was measured for the mixture of Q-PGEDA-OP/TPE derivative with constant concentration of TPE derivatives but varied concentrations of Q-PGEDA-OP. Progressive growth in the fluorescence
Figure 1. Hydrodynamic diameters and disrributions of (a) QPGEDA-OP and (c) Q-PGEDA-OP/TPE. Typical SEM images of (b) Q-PGEDA-OP and (d) Q-PGEDA-OP/TPE. Scale bars: 200 nm.
emission was observed for the mixture of Q-PGEDA-OP and TPE (Figure S7). These results verified the distinctive AIE function of TPE derivative. Fluorometric Bacteria Detection. Given that the antibacterial activity of Q-PGEDA-OP/TPE was postulated to initiate from the electrostatic association of cationic Q-PGEDAOP and anionic cell membrane of bacteria, the competing interaction of TPE derivative and anionic species in the cell membrane of bacteria to the cationic Q-PGEDA-OP could result in the release of the complexed TPE derivative.24−26 Hence, the AIE-functional TPE derivative could potentially serve as the probe for insight into the molecular basis of the antibacterial activity of the proposed Q-PGEDA-OP/TPE nanoparticles. To verify our assumption and explore the underlying antibacterial mechanism, the prepared Q-PGEDAOP/TPE (10 μM) was injected to the samples of AMOr S. aureus and AMOr E. coli (PBS was included as the blank control). Marked drop in fluorescence emission of TPE was affirmed for both AMOr S. aureus and AMOr E. coli (Figure 2), indicating the liberation of TPE from the Q-PGEDA-OP/TPE nanoparticles, affirming the possibility of structural dissociation of Q-PGEDA-OP/TPE nanoparticles as a consequence of electrostatic-based exchange reaction of anionic species in cell membrane of bacteria to Q-PGEDA-OP/TPE nanoparticles. Moreover, a consistent drop in fluorescence emission of TPE was observed for the reaction solution of bacteria and QPGEDA-OP/TPE nanoparticles along a rising population of bacteria, suggesting progressive occurrence of complex dissociation along the rising presence of bacteria. Relative to the bacteria-free sample, an approximate 50% loss in emission intensity can be observed post treatment with 109 CFU mL−1 of AMOr S. aureus or AMOr E. coli cells. Over 10% fluorescence intensity decrease can be recorded with the presence of a mere population (106 CFU mL−1) of AMOr S. aureus or AMOr E. coli. These results suggest the functional usage of the proposed system of Q-PGEDA-OP/TPE nanoparticles as the probe for determination of bacterial existence, which could be even possibly used as a tool for quantification of bacterial population provided a calibration was established (Scheme 1). Antimicrobial Activity. The antimicrobial activity of QPGEDA-OP/TPE was explored for treatment of AMOr S. D
DOI: 10.1021/acs.chemmater.8b00251 Chem. Mater. XXXX, XXX, XXX−XXX
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Figure 2. Fluorescence intensity measurement (λex = 330 nm) for the complex of Q-PGEDA-OP/TPE (10 μM, 20:1) in PBS with supplementation of AMOr S. aureus (left) and AMOr E. coli (right) with various concentrations (the numbers in the figure correspond to the concentration of bacterial with the unit of CFU mL−1). The insets are photographs of Q-PGEDA-TPE PBS with and without AMOr S. aureus or AMOr E. coli cells irradiated under a UV lamp (365 nm).
aureus or AMOr E. coli. The potency to the antibiotic-resistant bacteria was tested by the proposed Q-PGEDA-OP/TPE system. To obtained AMO-resistant bacteria, cultures were passaged 30 times in sequential treatment with rising concentrations of AMO.27 As summarized in Table 1 and
system exhibited appreciable antibacterial activeties (exceeding 99%) for treatment of both AMOr S. aureus and AMOr E. coli, which is attributable to the antimicrobial component of QA and strong interactions with bacteria. Q-PGEDA-OP/TPE was further explored by fluorescence microscopy for insight into the its antibacterial activities to AMOr S. aureus and AMOr E. coli. Herein, the bacteria with a compromised membrane were stained into red from ethidium bromide (EB) while the bacteria with intact membrane were stained into green from acridine orange (AO).27,32 After 30 min of treatment for AMOr S. aureus and AMOr E. coli with QPGEDA-OP/TPE, the untreated bacterial cells were confirmed to be uniform green fluorescence, in contrast to the largely red fluorescence, indicating the potent antibacterial activities of QPGEDA-OP/TPE (Figure S9). The results verified that QPGEDA-OP-treated bacteria could induce disruption to the membranes of bacteria, which is conducive to the passage of EB into the bacteria. Tailored Hydrolysis Behavior. Antimicrobial resistance arising from widespread of both rational use and abuse/misuse of germicides, complicated the situation of bacterial infectious disease.33−35 Indeed, a number of studies have reported the novel antibacterial systems in providing appreciable potency to treat antibiotic-resistant microorganisms.36−38 Nevertheless, an ideal microbial system capable of eliminating the antimicrobial resistance are seldom established,9,39 which should afford antibacterial potency to the targets for the limited period of time, followed by readily biodegradation into an inactive, nontoxic fragments. In the current reported antibacterial system, the structure of Q-PGEDA-OP could be readily converted by lipase, which is crucially important for minimizing the possibility of antimicrobial resistance and environmental issues. The antibacterial activity was strategically achieved by molecular design of Q-PGEDA-OP with cleavable ester linkage between antibacterial functional segments, namely, susceptible to hydrolysis into benign products, in a natural environment. Here, we confirmed the hydrolysis behavior of the tailored Q-PGEDA-OP antibacterial species. As documented, ester bond was subjected to complete cleavage in the presence of NaOH. Therefore, we attempted to treat Q-PGEDA-OP with 0.01 M aqueous NaOH at 50 °C overnight. The NaOH-treated products with carboxyl end-groups was determined to be Q-
Table 1. MICs of Antibiotics Prior to and Post Alkaline Treatment minimal inhibitory concentration (μg mL−1) antibiotics
AMOr S. aureus
AMOr E. coli
Q-PGEDA-OP/TPE NaOH-treated Q-PGEDA-OP/TPE Q-PGEDA-OA NaOH-treated Q-PGEDA-OA/TPE
15.5 >1000 31.5 31.5
125 >2000 500 500
Figure S8, the MIC of the proposed Q-PGEDA-OP/TPE was determined to be approximate 15.5 and 125 μg mL−1 for AMOr S. aureus and AMOr E. coli, respectively, whose MIC of 100 μg mL−1, even sub-100 μg mL−1, is appreciable for treatment of such robust bacteria strains. Note that the surface of Grampositive bacteria containing negatively charged teichoic acids and a thick peptidoglycan layer, is more readily able to bind to quaternary ammonium (QA) components than Gram-negative ones (composed of a lipopolysaccharide-linked external membrane). Hence, it is reasonable that more pronounced antibacterial potency was determined for AMOr S. aureus.28,29 The tempting antibacterial activity was speculated to be attributable to the potent interactions of QA with the cell membrane of bacteria, as well as the appropriate length of hydrophobic Q-PGEDA-OP side chain to insert into cell membranes.9,30,31 Interestingly, Q-PGEDA-OA, with only one quaternary ammonium salt group, exhibited limited antibacterial activity (MIC: 31.5 μg mL−1 and 500 μg mL−1 for AMOr S. aureus and AMOr E. coli), respectively. The time-dependent antibacterial performance of Q-PGEDA-OP/TPE was recorded for evaluation of the inhibition of bacteria growth. As depicted in Figure 3, AMO indicated relatively low inhibition for treatment of AMO-resistant bacteria (with 48.9% and 29.4% bacterial viability for AMOr S. aureus and AMOr E. coli, respectively). In contrast, the proposed Q-PGEDA-OP/TPE E
DOI: 10.1021/acs.chemmater.8b00251 Chem. Mater. XXXX, XXX, XXX−XXX
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Figure 3. (a) Photographs of colony forming units (CFU) for AMOr S. aureus and AMOr E. coli treated with AMO or Q-PGEDA-OP/TPE on the LB agar plate; CFU ratio of (b) AMOr S. aureus and (c) AMOr E. coli. Note: Asterisk (*) indicates significant differences (p < 0.05) between every two groups. Results are shown as mean ± standard deviation (n = 3).
Figure 4. SEM images of AMOr S. aureus (a−c) and AMOr E. coli (d−f) treated with PBS (a,d) and Q-PGEDA-OP (b,e) and Q-PGEDA-COOH (c,f). Scale bars: 200 nm.
PGEDA-COOH. The 1H NMR spectrum shows the decrease of the integral of −CH2− (at 4.1 ppm) adjacent to ester decreases and disappearance of the −CH2− peak (at 1.4 ppm) of octyl post alkaline hydrolysis (Figure S10). Moreover, the emergence of the peak at 2.3 ppm, assigned to the −CH2− COOH group (Figure S10, asterisk), confirmed the hydrolysis of Q-PGEDA-OP. The remnant of ester signals post NaOH treatment was attributed to the undegradable ester bond in the backbone of PGEDA (Figure S4). Antimicrobial Activity after Degradation. The antimicrobial activity of the degraded Q-PGEDA-COOH was tested for treatment of AMO-resistant bacteria. The MICs of NaOH-treated Q-PGEDA-OP/TPE was determined to be above 1000 μg mL−1 for AMOr S. aureus and above 2000 μg mL−1 for AMOr E. coli, respectively, confirming the inactiveness of degraded Q-PGEDA-COOH (Table 1, Figure S8). As described previously, the antibacterial activity of Q-PGEDAOP/TPE attributed to the following steps: a) cationic QPGEDA-OP strongly interacted with negative charge of bacterial membrane; b) the appropriate length of hydrophobic Q-PGEDA-OP side chain inserted into the bacteria mem-
branes; c) antibacterial outcome due to the bacteria membrane damaging/disruption. Here, Q-PGEDA-OP post hydrolysis, the additional carboxylic groups were believed to decrease the association to the bacterial cytoplasmic membrane due to electrostatic repulsion (Scheme 1).40,41 In the control experiment with Q-PGEDA-OA, the antibacterial potencies were determined to be same for Q-PGEDA-OA with and without treatment of NaOH (Table 1). These results indicated the possibility of creating an antibacterial system that can be deactivated by cleaving a single ester bond, which consequently prevent germicides unpredictable long-term effects. To gain further evidence of the antibacterial behavior of the proposed Q-PGEDA-OP/TPE, SEM was employed to visualize the morphological changes of bacteria (AMOr S. aureus and AMOr E. coli). For the untreated control bacteria (Figure 4a,d), clear edges and intact bodies of AMO-resistant bacteria were observed. The bacterial cell membranes appeared collapsed, split and deformed post incubation with Q-PGEDA-OP (Figure 4b,e), as opposed to Q-PGEDA-COOH-treated bacteria possessing the clear and intact shape (Figure 4c,f). These F
DOI: 10.1021/acs.chemmater.8b00251 Chem. Mater. XXXX, XXX, XXX−XXX
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Figure 5. (a) Illustration of the structural modification of Q-PGEDA-OP post enzymatically catalyzed hydrolysis. (b) 1H NMR spectra to record the hydrolysis of Q-PGEDA-OP in the presence of lipase. (c) Time-dependent hydrolysis rate of Q-PGEDA-OP in the presence of lipase.
treated Q-PGEDA-OP/TPE for Gram-negative AMOr E. coli also decreased (Table S2), which validated the deactivated antibacterial activity of Q-PGEDA-OP/TPE due to lipasemediated hydrolysis to resolve the chronic antibacterial impacts.
observations were consistent with the previous antibacterial behaviors. Lipase-Catalyzed Hydrolysis. The ester linkage tailored into the antibacterial Q-PGEDA-OP/TPE antibacterial system was determined to be a facile switch to quench its antibacterial activity. Herein the possibility of ester linkage cleavage in QPGEDA-OP was investigated in the biological milieu. Notably, the omnipresent lipase is the most abundant enzyme responsible for hydrolysis of hydrophobic ester derivatives. Herein, the hydrolysis experiment with lipase was performed by treating the Q-PGEDA-OP with lipase in phosphate-buffered D2O (pH 7) at 37 °C. In order to characterize the lipaseinduced cleavage of the Q-PGEDA-OP, we carried out 1H NMR measurement for this aforementioned reaction solution at varied incubation period. In resemblance of the 1H NMR spectrum of Q-PGEDA-COOH, consistent increase of 2.3 ppm peaks was observed for the Q-PGEDA-OP in the presence of lipase with extension of incubation, suggesting the appreciable degradation profile of the Q-PGEDA-OP (Figure 5b, Figure S11). The integral ratio of the signal at 2.3 ppm to that at 3.3 ppm of fully degraded sample by NaOH was defined as 100% of degradation. The degree of hydrolysis was determined by comparing the integral ratio of peak of 2.3 ppm to that of 3.3 ppm. The degradation curve implied that the ester underwent consistent hydrolysis (50% hydrolysis of Q-PGEDA-OP into Q-PGEDA-COOH at 9 days post incubation, Figure 5c). Furthermore, the MIC of the lipase-catalyzed Q-PGEDA-OP/ TPE was carried out to directly measure the impact of ester hydrolysis on the biocidal efficacy. At days 3 and 7 post incubation, the mixture of phosphate buffer and Q-PGEDAOP/TPE with or without lipase was tested for its antibacterial activity. The MIC values of Q-PGEDA-OP/TPE at days 3 and 7 with reaction of lipase were determined to be 31.5 and 62.5 μg mL−1 for AMOr S. aureus, respectively, while the control group without reaction of lipase retained full antibacterial efficacy (Table S1). The antibacterial efficacy of the lipase-
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CONCLUSIONS In conclusion, we have engineered a novel biodegradable antibacterial catiomer, functionalized with bacterial membrane anchoring and disruptive components for potent antibacterial activities against resistant bacteria and hydrolysis ester linkage for appreciable biodegradable characters. Hydrolysis of this antibacterial material under omnipresent lipase transforms its biocidal activity from being vigorous to inert, which averts chronic impact on bacteria, consequently minimizing the possibility of antimicrobial resistance and resolving the issues of bactericides due to widespread usage. Meanwhile, the antibacterial mechanism investigation (with the aids of AIE luminogens) revealed the engineered antibacterial molecule as a versatile approach for treatment of both AMO-resistant Grampositive and Gram-negative bacteria through electrostatic reaction with the anionic bacteria membrane. Moreover, the AIE luminogens could be employed as facile probe to detect the existence of bacteria. Therefore, the proposed system provided an important platform to engineer new generation of antibacterial materials, which could be developed further for pursuit of broad biomedical applications.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.8b00251. Materials and detailed experimental methods; synthesis and characterizations of poly(glycidyl methacrylate) (PGMA), aminated PGMA, Q-PGEDA-OA, and TPEG
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(COOH)4; 1H NMR of TPE derivatives and polymers; infrared spectra of the polymers; DLS, SEM, and fluorescence intensity measurement; and supplementary results of antimicrobial activity and hydrolysis behavior of the polymers (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hui Gao: 0000-0002-5009-9999 Author Contributions #
(S.C., Q.C.) These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was funded by the National Natural Science Foundation of China (Nos. 21674080, 21374079), the 131 Talents Program of Tianjin, the Leading Talents Program of the Tianjin Educational Committee, and Distinguished Professor of Tianjin, Training Project of Innovation Team of Colleges and Universities in Tianjin. Q.C. acknowledges the 586 funding support from the Fundamental Research Funds for the Central Universities [No. DUT17RC(3)059]. H.G. acknowledges the visiting professor program of Tsinghua University.
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