Conjugated Polymers Act Synergistically with Antibiotics to Combat

May 18, 2017 - The absorbance of each well at 600 nm was measured with a microplate reader and the antibacterial efficiency was calculated as describe...
37 downloads 10 Views 4MB Size
Download PDF
Research Article www.acsami.org

Conjugated Polymers Act Synergistically with Antibiotics to Combat Bacterial Drug Resistance Jingxiao Tian, Jiangyan Zhang, Jiangtao Yang, Lingyun Du, Hao Geng, and Yongqiang Cheng* Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei, P. R. China S Supporting Information *

ABSTRACT: The emergence of drug-resistant bacteria severely challenges the antimicrobial agents and antibacterial strategy. Here, we demonstrate a novel, simple, and highly efficient combination therapy strategy by direct combinations of cationic conjugated polymers (CCPs) with polypeptide antibiotics against Gram-negative and Gram-positive bacteria based on a synergistic antibacterial effect. The combination therapy method enhances the antibacterial efficacy with a significantly reduced antibiotic dosage. Also, the highly efficient and synergistic killing of drug-resistant bacteria is realized. Using combinations of CCPs and antibiotics to show increased antibacterial activity, this strategy will provide a much wider scope of the discovery of efficient antibacterial systems than that of antibiotic−antibiotic combinations. The proposed combination therapy method provides a universal and powerful platform for the treatment of pathogens, in particular, the drug-resistant bacteria, and also opens a new way for the development of efficient antibacterial systems. KEYWORDS: Cationic conjugated polymer, bacterial drug resistance, antibiotic, synergistic antimicrobial, combination therapy

■

INTRODUCTION Antibacterial antibiotics have been widely used in the treatment of infectious diseases of humans and animals. As humans use antibiotics frequently and irrationally, the problem of antibiotic resistance is increasingly highlighted.1,2 Thus, the drug-resistant bacteria have become the most serious pathogens of infectious diseases and also have brought great challenges to clinical treatment.3,4 With the increase in bacterial resistance, dosages of antibiotics also increase, leading to side effects to the human body. Although scientists are still trying to develop new antibiotics, more and more single-resistant and multiresistant bacteria would arise along with the results of bacteria against antibiotics. Now the rational use of antibiotics is one of the dominating therapy methods against pathogenic bacteria. Recently, many strategies based on new delivery materials have been developed to enhance the antibacterial efficacy of commonly used antibiotics and combat bacterial drug resistance,5 such as graphene,6,7 gold,8,9 and silver nanoparticles.10,11 Moreover, combination of nanometer materials and antibiotics were investigated to enhance the antibacterial efficiency via a synergistic mechanism.12−14 However, these metal nanomaterials have limitations as antibacterial agents, arising from their inherent toxicity and the characteristic of being difficult to degrade, which brings about a long-term retention in the body. Therefore, development of new materials and new strategies for rapid and efficient killing of bacteria attracts much more attention. In view of the less killing activity © 2017 American Chemical Society

of small dosage of single antibiotics, combinations of antibiotics are also used for the treatment of bacteria, especially drugresistant bacteria.15 The strategy of antibiotic combinations could broaden the antibacterial spectrum and generate synergistic effects. However, the discovery of effective antibiotic combinations has been limited in scope. Alternatively, several combinations of nonantibiotic molecules with antibiotics have shown increased antibacterial activity.16−18 This strategy will provide a much wider scope of discovery of an efficient antibacterial system than that of antibiotic combinations. Because of a delocalized electronic structure and unique light-harvesting features, conjugated polymers (CPs) have attracted much attention in biosensing, imaging, and biomedical applications.19−25 Therefore, many CPs have been designed and synthesized for antimicrobial application. The antibacterial mechanism of CPs is generally based on their unique phototoxicity and dark toxicity.26−29 Recently, Zhu et al. demonstrated that cationic conjugated polymers (CCPs) could bind to Escherichia coli through electrostatic and hydrophobic interactions and slightly loosen the bacterial membranes to enhance the release of intracellular contents of E. coli.30 In this work, we explore a new strategy based on a synergistic antibacterial mechanism of CCP−antibiotic combinations to Received: March 19, 2017 Accepted: May 18, 2017 Published: May 18, 2017 18512

DOI: 10.1021/acsami.7b03906 ACS Appl. Mater. Interfaces 2017, 9, 18512−18520

Research Article

ACS Applied Materials & Interfaces

where A was the absorbance of the bacteria control without adding CCPs or antibiotics and the other operations were identical to the experiment group, B was the absorbance of the LB culture medium itself as the background value, and C was the absorbance of the every experiment group. The MIC value of different samples was measured as the drug concentration that inhibited 90% of the bacteria growth. Effect on the Antibacterial Efficiency as a Function of the Incubation Time of PBF and PLB. Aliquot of 980 μL of the kanar E. coli solution at OD600 = 0.5 was transferred into a 24-well plate, in each well of which were added 4 μg/mL PLB, 10 μM PBF, and both PLB and PBF, respectively, to a final volume of 1 mL. Then, the 24-well plate was cultured for 0.5, 1, 2, and 3 h at 37 °C and 180 rpm in the dark. Subsequently, aliquot of 50 μL of the cultured solution from each well was transferred into a 48-well plate, followed by addition of 450 μL of LB medium. Each sample was performed in triplicate. The 48well plate was incubated at 37 °C and 180 rpm for 3 h. The absorbance of each well at 600 nm was measured with a microplate reader and the antibacterial efficiency was calculated as described above. Antibacterial Activity Test by Colony Counting. The above four kanar E. coli solutions in the 24-well plate, which respectively contained E. coli only, E. coli with 4 μg/mL PLB, 10 μM PBF, and both PLB and PBF to a final volume of 1 mL, were cultured for 2 h at 37 °C and 180 rpm in the dark. Afterward, the solutions were transferred to 1.5 mL tubes for centrifugation at 8000 rpm, respectively, for 2 min and then were washed with PBS buffer. After discarding the supernatant, all of the remaining kanar E. coli pellets were resuspended in PBS buffer and serially diluted 1 × 104 fold with PBS, respectively. Subsequently, a 100 μL portion of the bacterial dilution was spread on the solid LB agar plate and the colonies formed after 12 h incubation at 37 °C in the dark were counted. Each group of samples was performed through three parallel experiments. The killing efficiency (%) was calculated according to the following equation

enhance the antibacterial efficacy and efficiently treat drugresistant bacteria. By a combination of CCPs and antibiotics, the highly efficient synergistic killing of drug-resistant bacteria is realized. The proposed combination therapy method will provide a universal and powerful platform for the treatment of pathogens, in particular, the drug-resistant bacteria.

■

EXPERIMENTAL SECTION

Materials and Reagents. CCPs of a polyfluorene derivative (PBF) and a polythiophene derivative (PT) were prepared as described in the literature.31,32 Ampicillin (Amp), kanamycin sulfate, sulfamethizol (SMT), and gentamicin sulfate were purchased from Sigma. Polymyxin B (PLB) was purchased from Merck. Polymyxin E (PLE) was purchased from Yijishiye Co., Ltd. (Shanghai, China). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased from Beijing Xinjingke Biotechnology Co., Ltd. SYTOX Green nucleic acid stain was purchased from Molecular Probes (Eugene, OR). Polyethylenimine (PEI) was purchased from Alfa Aesar. The cell culture plates (24-well, 48-well, and 96-well) were obtained from Thermo Fisher. E. coli (BL21) and Bacillus subtilis were purchased from Beijing Bio-Med Technology Development Co., Ltd. The kanamycin-resistant (kanar) BL21 and green fluorescent protein (GFP) expressing kanar BL21 were obtained as previously described in the literature.30 Instruments. The optical densities of the bacteria at 600 nm were measured using a TU-1901 ultraviolet−visible spectrophotometer (Purkinje, China) and a microplate reader SpectraMax M2 (Molecular Devices). Fluorescence spectra were recorded on a FS5 fluorescence spectrometer (Edinburgh Instruments). ζ potentials were determined by a Nano ZS90 (Malvern, UK). Confocal laser scanning microscope (CLSM) experiments were performed with a FV1000-IX81 CLSM (Olympus). Scanning electron microscope (SEM) data were generated in the JSM-7500F SEM (JEOL). Plate counting photographs were taken by a VersaDoc imaging system (Bio-RAD). Bacterial Culture. In the assay, a kanar Gram-negative E. coli (BL21) was chosen as the research model. First, kanar BL21 picked from a single colony was transferred to 10 mL of the Luria-Bertani (LB) medium with kanamycin (50 μg/mL) then incubated in the dark at 37 °C and 180 rpm for about 4 h to an optical density of 0.5 at 600 nm (OD600 = 0.5). Next, we transferred the appropriate amount of bacterial solution for centrifugation at 8000 rpm for 2 min and discarded the supernatant. Then, the E. coli solution was washed with phosphate-buffered saline (PBS) buffer (1×) two times and suspended in it. ζ Potential Measurements. E. coli solutions (1 mL) treated with PBF, PLB, and PBF−PLB combination with OD600 = 0.5 were incubated at 37 °C for 2 h. After incubation, these were centrifuged at 8000 rpm for 3 min and then the precipitated pellets were washed with PBS buffer (1×) two times. Finally, the pellets were resuspended in ddH2O for ζ potential measurements. Exactly the same procedures were performed for the untreated E. coli solution (without PBF or PLB) as a negative control. Minimal Inhibitory Concentration (MIC) Determination of Antibiotics, CCPs, and CCP−Antibiotic Combinations Against Drug-Resistant E. coli. Aliquot of 180 μL of kanar BL21 at a density of 1.25 × 106 colony forming units (CFU)/well in LB medium was added into a 96-well plate. Next, different concentrations of antibiotics, CCPs, and both antibiotic and CCP were introduced into the medium, and the antibiotic was serially diluted two-fold by LB solution. The final volume in each well was 200 μL. The bacterial solution was incubated in the dark at 37 °C and 180 rpm for 12 h. Finally, we measured the absorbance at 600 nm with a microplate reader and calculated the antibacterial efficiency. The antibacterial efficiency (%) was calculated according to the following equation

antibacterial efficiency (%) = (S0 − S)/ S0 × 100% where S is the CFU of the experimental group treated with PBF, PLB alone, and in combination with PBF and PLB, respectively, and S0 is the CFU of the bare E. coli only, without any treatment. Protein Release Test and CLSM and SEM Characterization of E. coli. A single colony of GFP-expressing E. coli BL21 (BL21-GFP) was transferred to 10 mL of the LB/kanar culture medium and incubated at 37 °C and 180 rpm for 2 h to an OD600 of about 0.4. Afterward, we added 10 μL of IPTG (25 mg/mL) to the culture medium and continued to culture it for about 4 h to an OD600 of about 1.0. Next, the solution was transferred and centrifuged at 8000 rpm for 3 min, washed with PBS (1×) three times, and diluted with PBS (1×) to an OD600 of about 0.5. Subsequently, we treated the bacteria with PLB (4 μg/mL), PBF (10 μM), and a combination of PLB and PBF, respectively, in 1 mL PBS buffer at 37 °C and 180 rpm for 2 h and centrifuged it to obtain the supernatant and bacterial pellets. Finally, the supernatant was used to measure the fluorescence spectrum. The BL21-GFP bacterial pellets were suspended in PBS buffer for CLSM and SEM imaging. For CLSM imaging, 10 μL of the BL21-GFP suspension was transferred to a glass slide for imaging. For SEM imaging, the BL21-GFP suspensions were collected and immediately fixed with glutaraldehyde (0.5%) in PBS buffer at room temperature for 30 min. Afterward, the bacterial suspensions were centrifuged at 8000 rpm for 2 min and the supernatant was removed and then the bacterial pellets were suspended in sterile water. Next, 3 μL of bacterial suspensions was added to clean silicon slices followed by naturally drying in the air. Then, 0.1% glutaraldehyde in PBS buffer was added for fixation for 1 h and then 0.5% glutaraldehyde overnight. Next, the specimens were washed with sterile water two times and dehydrated by the addition of ethanol in a graded series of 60, 70, 90, and 100% for 6 min, respectively, and then dried. Finally, the specimens were coated with platinum before SEM measurements.

antibacterial efficiency (%) = {[(A − B) − (C − B)]/(A − B)} × 100% 18513

DOI: 10.1021/acsami.7b03906 ACS Appl. Mater. Interfaces 2017, 9, 18512−18520

Research Article

ACS Applied Materials & Interfaces

Scheme 1. Schematic of the Synergistic Antibacterial Mechanism of Combinations of a CCP with an Antibiotica

(a) bacteria, (b) bacteria + CCP, (c) bacteria + antibiotic, and (d) bacteria + CCP + antibiotic. “1 + 1 > 2” means that the efficiency of the combination of CCP with antibiotic for killing bacteria is much higher than the sum of that of the antibiotic and CCP used alone.

a

Scheme 2. Chemical Structures of the CCPs and Antibiotics Used in This Work

■

RESULTS AND DISCUSSION

membrane of bacteria treated with CCP may be slightly loose and coarse owing to the tight coating of CCP. Some antibiotics, such as polypeptide antibiotics,33 can alter bacterial outer membrane permeability by binding to bacteria through electrostatic interactions, resulting in the leakage of intracellular contents and bacterial death. However, as shown in Scheme 1c, low dosage of a polypeptide antibiotic has little effect on bacteria, whose cell membrane structure is almost intact. For drug-resistant bacteria, even high dosage of antibiotic cannot

The schematic diagram of the synergistic antibacterial action by combinations of a CCP with an antibiotic is shown in Scheme 1. The CCP can generally bind to the surface of bacteria such as E. coli through electrostatic and hydrophobic interactions (Scheme 1b). Under dark conditions, the CCP itself exhibits low toxicity to bacteria. However, compared to the compact membrane structure of the bare bacteria (Scheme 1a), the cell 18514

DOI: 10.1021/acsami.7b03906 ACS Appl. Mater. Interfaces 2017, 9, 18512−18520

Research Article

ACS Applied Materials & Interfaces

μM for SMT and 30 μM for Amp), the combinations of PBF and Amp or SMT do not exhibit synergistically enhanced antibacterial effect. For kanar E. coli treated with PLB alone, as shown in Figure 1e, the antibacterial efficiency is enhanced from 10.2 to 99.7% upon increasing the PLB concentration from 0.5 to 4.0 μg/mL. The MIC value was determined to be about 3.7 μg/mL (Table 2). In comparison, by combining PLB with cationic polymer PBF, the antibacterial efficiency is obviously enhanced to 98.6% at a low PLB concentration of 2.5 μg/mL (antibacterial efficiency is only 25.8% for PLB alone, Figure 1e). The MIC value for a combination of PBF and PLB is measured to be about 2.3 μg/mL, which is much lower than that for PLB alone (Table 2). PLE, which is another polymyxin antibiotic with a similar antibacterial mechanism to that of PLB, also exhibits a distinctly enhanced antibacterial activity upon combining with PBF with a much lowered MIC value of 2.5 μg/mL in comparison to 15.1 μg/mL for PLE alone (Table 2). Actually, under the MIC dosage condition, the antibacterial efficiency for combinations of PBF and PLE is much higher than the sum of that of PBF and PLE alone (90% against 2.1% + 7.2% = 9.3%, Figure 1f). These results indicate that the unexpected synergistic combinations of cationic polymer PBF and low dosage of polymyxin antibiotics exhibit significantly enhanced antibacterial activity toward drug-resistant bacteria through the outer membrane relaxation mechanism. The antibacterial activity of PBF and PLB alone and the synergistic antibacterial effect of PBF−PLB combinations toward kanar E. coli as a function of incubation time were investigated. As shown in Figure 2a, the antibacterial efficiencies of PBF and PLB when used alone are low and slightly increased with the extension of incubation time from 0.5 to 3 h. In comparison with PBF and PLB alone, under the optimized concentrations of PBF and PLB ([PLB] = 4 μg/mL, [PBF] = 10 μM; Supporting Information, Figure S1), combinations of PBF and PLB exhibit much higher antibacterial efficiencies that increase from 78 up to 98% with the extension of incubation time from 0.5 to 2 h. As shown in Figure 2b, the colony counting results also show that the antibacterial efficiencies of PBF, PLB, and PBF−PLB combinations are 9.5, 10.4, and 92.5%, respectively. These results indicate that the enhanced antibacterial activity of PBF−PLB combinations originates from their synergistic effect. To verify the mechanism of the synergistic antibacterial effect of PBF−PLB combinations, a protein releasing experiment was performed by treatments of GFP-expressing kanar E. coli BL21 (E. coli-GFP) with PBF, PLB, and PBF−PLB combinations, respectively. When the cell membrane of E. coli-GFP is broken, the GFP will be released into the solution; thus, the higher the antibacterial activity of the antibiotic, the more the release of GFP. In these experiments, we incubated E. coli-GFP with PBF, PLB, and PBF−PLB combinations, respectively. The treated E. coli solutions were centrifuged, and the supernatants were collected for fluorescence measurements. As shown in Figure 2c, the fluorescence signal of GFP from the supernatant treated with cationic polymer PBF alone is similar to that from E. coli without any treatment, indicating that the membrane-breaking ability of PBF alone is low. Upon treatment of E. coli-GFP with 4 μg/mL PLB, because the binding of PLB leads to increased membrane permeability and thereby the leakage of intracellular contents, the fluorescence intensity of GFP is higher than that upon treatment with PBF. However, a much higher fluorescence intensity of GFP from the E. coli treated with PBF−PLB combinations was observed than that from E. coli

kill the bacteria. Although CCP or antibiotic alone has poor antibacterial activity, combinations of CCPs with antibiotics may achieve high antibacterial efficacy, originating from their synergistic membrane-disrupting action, thereby gaining one plus one is greater than two effects (“1 + 1 > 2”) (Scheme 1d). PBF and PT were chosen as CCPs in this work. To verify the synergistic antibacterial mechanism, we systematically explored combinations of the two CCPs with a collection of commonly used antibiotics including gentamicin (Gent), SMT, Amp, PLB, PLE, and the polypeptide model amphipathic peptide (MAP) (see the chemical structures of CCPs and antibiotics in Scheme 2). Gram-negative kanar E. coli type BL21 was chosen as the model of drug-resistant bacteria in this study. First, we investigated the interaction between CCP or antibiotic and E. coli by measuring the ζ potential of the E. coli surface. PBF that possesses multiple positive charges on the side chain and a hydrophobic fluorene and boron-dipyrromethene backbone can bind to the E. coli surface through electrostatic and hydrophobic interactions.31 PLB, a class of cationic and cyclic polypeptide antibiotics, is usually apt to bind to Gram-negative bacteria and disorganizes the bacterial membranes through a detergent-like mode of action.33 Therefore, ζ potential measurements were performed by studying the interactions of PBF with E. coli, PLB with E. coli, and PBF with PLB and E. coli. As shown in Table 1, the ζ potential of E. coli is measured to be Table 1. ζ Potential of E. coli Treated with PBF, PLB, and the PBF−PLB Combination ζ potential (mV) E. E. E. E.

coli coli + PBF coli + PLB coli + PBF + PLB

−43.38 −33.46 −35.64 −26.68

± ± ± ±

0.87 0.90 0.82 1.24

−43.38 ± 0.87 mV due to the negatively charged surface of E. coli. Upon addition of PBF or PLB alone, the ζ potential of E. coli exhibits a positive shift from −43.38 ± 0.87 mV to −33.46 ± 0.90 mV for PBF and −35.64 ± 0.82 for PLB, indicating that PBF or PLB alone is bound to the surface of E. coli. When PBF along with PLB is introduced in the E. coli solution, the ζ potential of E. coli became 26.68 ± 1.24, which is more positive than that from binding of PBF or PLB alone to E. coli. The results suggested that PBF and PLB could successfully bind to the surface of E. coli simultaneously, which provided the foundation for their synergistic antibacterial study. The synergistic antibacterial activity is systematically investigated by MIC assay. MIC is the lowest concentration of antimicrobial agents that will inhibit the growth of bacteria and is also the gold standard for the evaluation of the antimicrobial activity and potency of antimicrobial agents. As shown in Figure 1a, the antibacterial efficiency of PBF itself at 2.5 μM is about 3.2% and increases slowly with an increase in the concentration of PBF from 2.5 to 20 μM in the dark. Even up to 80 μM, its antibacterial efficiency reaches only 20%, suggesting that the antibacterial activity of PBF alone is low to kanar E. coli. Figure 1b shows that Gent and a combination of PBF and Gent also exhibit low antibacterial activity to kanar E. coli, which is because Gent and kanamycin belong to the same class of antibiotic and also kanar E. coli is resistant to Gent. Although antibiotics SMT and Amp, as shown in Figure 1c,d, exhibit above 80% antibacterial efficiency on a high dosage (250 18515

DOI: 10.1021/acsami.7b03906 ACS Appl. Mater. Interfaces 2017, 9, 18512−18520

Research Article

ACS Applied Materials & Interfaces

Figure 1. Antibacterial activities of PBF and antibiotics alone and PBF−antibiotic combinations toward kanar E. coli. (a) PBF alone; [PBF] = 2.5, 5, 10, 20, 40, and 80 μM. (b) Gent alone and combinations of Gent and PBF; [Gent] = 2, 4, 8, 16, 32, 64, and 128 μg/mL, [PBF] = 10 μM. (c) SMT alone and combinations of SMT and PBF; [SMT] = 2, 4, 8, 16, 32, 64, 128, and 256 μg/mL, [PBF] = 10 μM. (d) Amp alone and combinations of Amp with PBF; [Amp] = 1, 2, 4, 8, 16, 32, and 64 μg/mL, [PBF] = 10 μM. (e) PLB alone and combinations of PLB and PBF; [PLB] = 0.5, 1, 2, 2.5, 3, 3.5, 4, and 8 μg/mL, [PBF] = 10 μM. (f) PLE alone and combinations of PLE and PBF; [PLE] = 0.5, 1, 2, 4, 8, 16, and 32 μg/mL, [PBF] = 10 μM. Error bars were obtained from three replicate measurements.

indicate that PBF itself does not break the cell membrane to release the intracellular GFP. For the E. coli-GFP treated with PLB, despite the fact that PLB is an excellent antibiotic for E. coli with the membrane-disturbing feature, a small dosage of PLB ([PLB] = 4 μg/mL) is not enough to kill E. coli-GFP. Thus, E. coli morphology is not changed and GFP is retained in E. coli (Figure 3A3). However, for the E. coli-GFP treated with PBF−PLB combinations, intense aggregation of E. coli-GFP and a clearly cracked and collapsed morphology are observed, leading to the release of GFP. Therefore, no GFP fluorescence is observed in the fluorescence field, while PBF keeps binding to the cell membrane, and red fluorescence of PBF is still there (Figure 3A4). These results match well with those of the protein release experiment and further demonstrate the synergistic effect of PBF−PLB combinations for enhancing the antibacterial activity. To confirm the membrane-disturbing mechanism of the PBF−PLB combination, SYTOX Green stain, which can penetrate cells with compromised plasma membranes and will not cross the membranes of live cells, was utilized to stain E. coli for CLSM. As shown in Figure S2b,c, when PBF or PLB alone is incubated with E. coli, no green fluorescence from SYTOX Green dye is observed, indicating that the membranes of E. coli cells are not damaged under the treatment of PBF or PLB for the experimental dosage. Red fluorescence of PBF is acquired from the E. coli treated with PBF or PBF−PLB combination (Figure S2b,d), stemming from the binding interaction between PBF and E. coli. Nevertheless, the E. coli treated with the PBF− PLB combination shows the bright green fluorescence from SYTOX Green, indicating that the membrane of E. coli is

Table 2. MIC Values (μg/mL) of the Antibiotics for Treatment of Bacteria in the Presence and Absence of CCPsa antibiotic bacteria E. coli B. subtilis a

− PBF + PBF − PBF + PBF

PLB

PLE

MAP

Amp

Gent

SMT

3.7 2.3 7.5 3.1

15.1 2.5

3.8 1.9 0.45 0.23

32 32 0.64 0.48

>125 >125

>256 >256

Note: All of the MIC values are taken from Figures 1, S3, and S4.

treated with PBF or PLB. Given that PBF itself has little contribution to the increase of fluorescence intensity, it is reasonably proposed that the enhanced GFP release origins from the synergistic effect of PBF and PLB. To further get more insight into the synergistic antibacterial mechanism, CLSM imaging was used to directly observe the antibacterial effect of PBF−PLB combinations. The treated E. coli-GFP was centrifuged and subsequently suspended in PBS buffer for CLSM imaging. As shown in Figure 3A1, the E. coliGFP without any treatment exhibits extremely intact morphology in bright field and sufficient GFP expression in green fluorescence field. Upon incubation with PBF, although E. coli-GFP appears to aggregate as a result of the electrostatic interaction between the positively-charged PBF and negatively charged E. coli, the shape of E. coli-GFP is not changed and the GFP expression is not affected. Meanwhile, red fluorescence from PBF is observed on E. coli (Figure 3A2). These results 18516

DOI: 10.1021/acsami.7b03906 ACS Appl. Mater. Interfaces 2017, 9, 18512−18520

Research Article

ACS Applied Materials & Interfaces

Figure 2. (a) Antibacterial activities of PBF and PLB and the synergistic antibacterial effect of PBF−PLB combinations toward kanar E. coli as a function of incubation time. Error bars were obtained from three replicate measurements. (b) CFU for kanar E. coli without treatment (1) and when treated with PBF (2), PLB (3), and PBF−PLB combinations (4) on LB agar plates. (c) Fluorescence intensities of the released GFP from GFPexpressing kanar E. coli without treatment (1) and when treated with PBF (2), PLB (3), and PBF−PLB combinations (4). [PLB] = 4 μg/mL, [PBF] = 10 μM.

from 0.1 to 0.5 μg/mL. This result indicates that Amp itself possesses high antibacterial activity against B. subtilis. However, upon treatment of B. subtilis with PBF−Amp combinations, the synergistic antibacterial effect is not obvious. Hereafter, we tested the treatment of B. subtilis with PBF−PLB combinations. As shown in Figure S3c, PLB alone against B. subtilis shows a low antibacterial efficiency of about 10.8% with an increase in concentration from 0.5 to 4.0 μg/mL and the MIC value is measured to be 7.5 μg/mL (Table 2). However, it is expected that the antibacterial efficiency is remarkably enhanced to 97.8% with an increase in concentration of PLB from 0.5 to 4.0 μg/mL by PBF−PLB combinations ([PBF] = 10 μM) and the MIC value is reduced to 3.1 μg/mL (Table 2) using drug combinations. The antibacterial mechanism can be ascribed to the fact that PBF can make the cell wall of B. subtilis loosen, which facilitates PLB to access and damage the membrane structure, whereas PLB alone cannot make it. This result is interesting to extend the application of polypeptide antibiotics and provides a new therapeutic method for Gram-positive bacteria, indicating that the combination therapy using CCPs and polypeptides is effective toward both Gram-negative and Gram-positive bacteria. To explore the universality of this combination therapy method based on CCPs, we employed a MAP for evaluating the synergistic antibacterial effect. The MAP is an artificial cellpenetrating peptide with antibacterial activity.34,35 As shown in Figure S4a,b and Table 2, both MIC values for PBF−MAP combinations against E. coli and B. subtilis are significantly reduced as compared to those for MAP alone, indicating that the combination of CCP with antibacterial peptides exhibits a good synergistic antibacterial effect. This is of instructive significance to synthesize and develop new antibiotics for combination therapy as well as killing drug-resistant bacteria.

damaged by the PBF−PLB combination. These results also provide direct evidence for the synergistic antibacterial effect of the PBF−PLB combination with a membrane-disturbing mechanism. To further investigate the synergistic antibacterial mechanism, the microscopic structure of E. coli treated with PBF− PLB combinations was studied by SEM. The morphology of the untreated E. coli is intact, and the membrane structure of E. coli is smooth (Figure 3B1). For the E. coli treated with only PBF, the membrane surface is slightly deformed and becomes coarse (Figure 3B2). These results may be attributed to the fact that the tight coating and destructive intercalation of PBF into the membranes cause the bacterial membranes relaxation. For the E. coli treated with only PLB, because the dosage of PLB of 4 μg/mL is low and nonlethal, the morphology and membrane structure remain intact (Figure 3B3). In contrast, the E. coli treated with PBF−PLB combinations show collapsed, ruptured, and fused morphologies (Figure 3B4), which further confirm the synergistic antibacterial effect of PBF−PLB combinations. To explore the applicability of the combination therapy, the synergistic antibacterial effects toward Gram-positive bacteria, B. subtilis, were investigated by combining cationic polymer PBF with antibiotics Amp and PLB, respectively. As shown in Figure S3a, the antibacterial efficiency of PBF alone against B. subtilis is low (about 10%) in the range of 2.5−10 μM and is significantly enhanced up to 96.2% with an increase in the concentration of PBF from 10 to 40 μM. Therefore, 10 μM PBF was selected to combine with the antibiotics to investigate the synergistic antibacterial activity. Because of the high antibacterial activity of antibiotic Amp against Gram-positive bacteria, it was first chosen to combine with PBF for evaluating the synergistic antibacterial effect on B. subtilis. Figure S3b shows that the antibacterial efficiency of Amp alone increases obviously up to 87.4% with an increase in the concentration 18517

DOI: 10.1021/acsami.7b03906 ACS Appl. Mater. Interfaces 2017, 9, 18512−18520

Research Article

ACS Applied Materials & Interfaces

Figure 3. (A) CLSM imaging of GFP-expressing kanar E. coli without treatment (1) and when treated with PBF (2), PLB (3), and PBF−PLB combinations (4); [PLB] = 4 μg/mL, [PBF] = 10 μM. (B) SEM characterization of the E. coli: (1) E. coli, (2) E. coli + PBF, (3) E. coli + PLB, (4) E. coli + PBF−PLB combination.

efficiency of the PEI−PLB combination toward E. coli is lower than the sum of that of PEI and PLB alone (Figure S4f). This result suggests that the combination of the nonconjugated polyelectrolyte and PLB has no the synergistic antibacterial effect. We infer that the conjugated backbone structure of polyelectrolytes contributes toward the synergistic antibacterial effect of the combination of the CCP and polypeptide antibiotic. For the combination therapy, it is important to select the combinative agents and their doses for highly efficient bacterial killing. In this study, we selected CCP−antibiotic combinations for studying their broad-spectrum antibacterial activity, where CCPs are easy to coat the surface of bacteria through electrostatic and hydrophobic interactions. Among the antibiotics, PLB is very suitable for the combination therapy study considering its characteristic features that alter bacterial outer membrane permeability and leak cellular molecules. We have successfully demonstrated that combinations of low doses of

Next, we evaluated the synergistic antibacterial effect of the combination of PLB and another CCP, PT. Figure S4c,d shows that the antibacterial efficiency of PT alone is very low toward E. coli in the concentration range of 2.5−80 μM. Upon combining 2.5 μM PT with PLB, the MIC value toward E. coli is 1.2 μg/mL, which is much lower than that of PLB alone (MIC: 3.7 μg/mL). These results show that the proposed synergistic antibacterial therapy is suitable for various polypeptide drugs and CCPs, exhibiting extensive universality. Meanwhile, to investigate whether the synergistic antibacterial effect is related to the conjugated backbone structure of CCP, a control experiment using a nonconjugated polyelectrolyte, PEI, was performed with PLB. Figure S4e shows that the antibacterial efficiency of PEI toward E. coli increases with increasing concentration of PEI from 1 to 128 μg/mL and exhibits above 80% antibacterial efficiency at 128 μg/mL. However, when 8 μg/mL PEI (25.6% killing efficiency itself toward E. coli) is used in combination with PLB, the killing 18518

DOI: 10.1021/acsami.7b03906 ACS Appl. Mater. Interfaces 2017, 9, 18512−18520

ACS Applied Materials & Interfaces cationic polymer PBF and PLB exhibit a high synergistic killing activity against kanar E. coli, using MIC testing as the gold standard for evaluating the antibiotic potency, whereas PBF or PLB alone possesses poor antibacterial activity. The mechanism of the synergistic antibacterial effect was investigated systemically by testing the protein released from E. coli and by studying the changes in the morphology and microscopic structure by the CLSM and SEM techniques. As compared with the intact morphology and structure of the E. coli treated with PLB or PBF alone, PBF and PLB combinations showed a significant rupture and collapse on the surface of E. coli. Because PBF alone can tightly coat, intercalate into, and relax the membranes of bacteria, we infer that the PBF and PLB combination further facilitates and accelerates the rupture and collapse of bacterial membranes, which results in the release of intracellular contents and bacterial death. What is more, a similar synergistic antibacterial result was acquired by the combination of PBF with another polymyxin antibiotic PLE. However, some other kind of antibiotics without membrane-disturbing ability, such as Amp and SMT, showed noneffectively synergistic antibacterial activity. These results demonstrated that the polypeptide antibiotics used to combine with the cationic polymer for synergistic therapy are special because of their unique antibacterial features.

ACKNOWLEDGMENTS

■

REFERENCES

(1) Li, J.; Nation, R. L.; Turnidge, J. D.; Milne, R. W.; Coulthard, K.; Rayner, C. R.; Paterson, D. L. Colistin: The Re-emerging Antibiotic for Multidrug-resistant Gram-negative Bacterial Infections. Lancet Infect. Dis. 2006, 6, 589−601. (2) Taubes, G. The Bacteria Fight Back. Science 2008, 321, 356−361. (3) Fischbach, M. A.; Walsh, C. T. Antibiotics for Emerging Pathogens. Science 2009, 325, 1089−1093. (4) Waldron, K. J.; Robinson, N. J. How Do Bacterial Cells Ensure That Metalloproteins Get the Correct Metal? Nat. Rev. Microbiol. 2009, 7, 25−35. (5) Pelgrift, R. Y.; Friedman, A. J. Nanotechnology As A Therapeutic Tool to Combat Microbial Resistance. Adv. Drug Delivery Rev. 2013, 65, 1803−1815. (6) Perreault, F.; Faria, A. F.; Nejati, S.; Elimelech, M. Antimicrobial Properties of Graphene Oxide Nanosheets: Why Size Matters. ACS Nano 2015, 9, 7226−7236. (7) Qi, Z.; Bharate, P.; Lai, C. H.; Ziem, B.; Bottcher, C.; Schulz, A.; Beckert, F.; Hatting, B.; Mulhaupt, R.; Seeberger, P. H.; Haag, R. Multivalency at Interfaces: Supramolecular Carbohydrate-functionalized Graphene Derivatives for Bacterial Capture, Release, and Disinfection. Nano Lett. 2015, 15, 6051−6057. (8) Zhao, Y.; Chen, Z.; Chen, Y.; Xu, J.; Li, J.; Jiang, X. Synergy of Non-antibiotic Drugs and Pyrimidinethiol on Gold Nanoparticles Against Superbugs. J. Am. Chem. Soc. 2013, 135, 12940−12943. (9) Li, X.; Robinson, S. M.; Gupta, A.; Saha, K.; Jiang, Z.; Moyano, D. F.; Sahar, A.; Riley, M. A.; Rotello, V. M. Functional Gold Nanoparticles as Potent Antimicrobial Agents Against Multi-drugresistant Bacteria. ACS Nano 2014, 8, 10682−10686. (10) Chernousova, S.; Epple, M. Silver as Antibacterial Agent: Ion, Nanoparticle, and Metal. Angew. Chem., Int. Ed. 2013, 52, 1636−1653. (11) Cochis, A.; Azzimonti, B.; Valle, C. D.; Giglio, E. D.; Bloise, N.; Visai, L.; Cometa, S.; Rimondini, L.; Chiesa, R. The Effect of Silver or Gallium Doped Titanium Against the Multidrug Resistant Acinetobacter Baumannii. Biomaterials 2016, 80, 80−95. (12) Morones-Ramirez, J. R.; Winkler, J. A.; Spina, C. S.; Collins, J. J. Silver Enhances Antibiotic Activity Against Gram-negative Bacteria. Sci. Transl. Med. 2013, 5, 190ra81. (13) Richter, A. P.; Brown, J. S.; Bharti, B.; Wang, A.; Gangwal, S.; Houck, K.; Hubal, E. A. C.; Paunov, V. N.; Stoyanov, S. D.; Velev, O. D. An Environmentally Benign Antimicrobial Nanoparticle Based on A Silver-infused Lignin Core. Nat. Nanotechnol. 2015, 10, 817−823. (14) Zhao, Y.; Cao, H.; Qin, H.; Cheng, T.; Qian, S.; Cheng, M.; Peng, X.; Wang, J.; Zhang, Y.; Jin, G.; Zhang, X.; Liu, X.; Chu, P. K. Balancing the Osteogenic and Antibacterial Properties of Titanium by Codoping of Mg and Ag: An in Vitro and in Vivo Study. ACS Appl. Mater. Interfaces 2015, 7, 17826−17836. (15) Walsh, C. Molecular Mechanisms That Confer Antibacterial Drug Resistance. Nature 2000, 406, 775−781. (16) Li, W.; Dong, K.; Ren, J.; Qu, X. A β-Lactamase-Imprinted Responsive Hydrogel for the Treatment of Antibiotic-Resistant Bacteria. Angew. Chem., Int. Ed. 2016, 55, 8049−8053. (17) Mazumdar, K.; Dastidar, S. G.; Park, J. H.; Dutta, N. K. The Anti-inflammatory Non-antibiotic Helper Compound Diclofenac: An Antibacterial Drug Target. Eur. J. Clin. Microbiol. Infect. Dis. 2009, 28, 881−891. (18) Ejim, L.; Farha, M. A.; Falconer, S. B.; Wildenhain, J.; Coombes, B. K.; Tyers, M.; Brown, E. D.; Wright, G. D. Combinations of Antibiotics and Nonantibiotic Drugs Enhance Antimicrobial Efficay. Nat. Chem. Biol. 2011, 7, 348−350.

CONCLUSIONS In summary, we demonstrate a novel, simple, and highly efficient combination therapy strategy by directly combining CCP with polypeptide antibiotics against Gram-negative and Gram-positive bacteria. The antibacterial efficiency is improved due to the synergistic antibacterial mechanism of the combination therapy. The combination therapy method can enhance the antibacterial efficacy with a significantly reduced antibiotic dosage, and the highly efficient synergistical killing of drug-resistant bacteria is also realized. Using combinations of cationic polymers with antibiotics to show increased antibacterial activity, this strategy will provide a much wider scope of discovery of efficient antibacterial systems than that of antibiotic−antibiotic combinations. The proposed combination therapy method will provide a universal and powerful platform for the treatment of pathogens, in particular, the drug-resistant bacteria. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b03906. Optimization of experimental conditions and additional Figures S1−S4 (PDF)

■

■

The authors are grateful for financial support of the National Natural Science Foundation of China (Nos. 21475031 and 21605034) and the National Science Foundation of Hebei Province (B2016201031).

■

■

Research Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: +86-312-5079403. ORCID

Yongqiang Cheng: 0000-0002-9569-9517 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. 18519

DOI: 10.1021/acsami.7b03906 ACS Appl. Mater. Interfaces 2017, 9, 18512−18520

Research Article

ACS Applied Materials & Interfaces (19) Feng, X.; Liu, L.; Yang, Q.; Wang, S. Water-soluble Fluorescent Conjugated Polymers and Their Interactions with Biomacromolecules for Sensitive Biosensors. Chem. Soc. Rev. 2010, 39, 2411−2419. (20) Zhu, C.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. Water-soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy. Chem. Rev. 2012, 112, 4687−4735. (21) Disney, M. D.; Zheng, J.; Swager, T. M.; Seeberger, P. H. Detection of Bacteria with Carbohydrate-functionalized Fluorescent Polymers. J. Am. Chem. Soc. 2004, 126, 13343−13346. (22) Phillips, R. L.; Kim, I. B.; Carson, B. E.; Tidbeck, B.; Bai, Y.; Lowary, T. L.; Tolbert, L. M.; Bunz, U. H. F. Sugar-substituted Poly(pphenyleneethynylene)s: Sensitivity Enhancement Toward Lectins and Bacteria. Macromolecules 2008, 41, 7316−7320. (23) Nederberg, F.; Zhang, Y.; Tan, J. P. K.; Xu, K.; Wang, H.; Yang, C.; Gao, S.; Guo, X. D.; Fukushima, K.; Li, L.; Hedrick, J. L.; Yang, Y. Y. Biodegradable Nanostructures with Selective Lysis of Microbial Membranes. Nat. Chem. 2011, 3, 409−414. (24) Wang, Y.; Chi, E. Y.; Natvig, D. O.; Schanze, K. S.; Whitten, D. G. Antimicrobial Activity of Cationic Conjugated Polyelectrolytes and Oligomers Against Saccharomyces Cerevisiae Vegetative Cells and Ascospores. ACS Appl. Mater. Interfaces 2013, 5, 4555−4561. (25) Yuan, H.; Wang, B.; Lv, F.; Liu, L.; Wang, S. Conjugated Polymer-based Energy Transfer Systems for Antimicrobial and Anticancer. Adv. Mater. 2014, 26, 6978−6982. (26) Tang, Y. L.; Corbitt, T. S.; Parthasarathy, A.; Zhou, Z.; Schanze, K. S.; Whitten, D. G. Light-induced Antibacterial Activity of Symmetrical and Asymmetrical Oligophenylene Ethynylenes. Langmuir 2011, 27, 4956−4962. (27) Corbitt, T. S.; Ding, L. P.; Ji, E.; Ista, L. K.; Ogawa, K.; Lopez, G. P.; Schanze, K. S.; Whitten, D. G. Light and Dark Biocidal Activity of Cationic Poly(arylene ethynylene) Conjugated Polyelectrolytes. Photochem. Photobiol. Sci. 2009, 8, 998−1005. (28) Wang, Y.; Jett, S. D.; Crum, J.; Schanze, K. S.; Chi, E. Y.; Whitten, D. G. Understanding the Dark and Light-enhanced Bactericidal Action of Cationic Conjugated Polyelectrolytes and Oligomers. Langmuir 2013, 29, 781−792. (29) Kenawy, E.-R.; Worley, S. D.; Broughton, R. The Chemistry and Applications of Antimicrobial Polymers: A State-of-the-art Review. Biomacromolecules 2007, 8, 1359−1384. (30) Zhu, C.; Yang, Q.; Lv, F.; Liu, L.; Wang, S. Conjugated Polymer-coated Bacteria for Multimodal Intracellular and Extracellular Anticancer Activity. Adv. Mater. 2013, 25, 1203−1208. (31) Chong, H.; Nie, C. Y.; Zhu, C. L.; Yang, Q.; Liu, L. B.; Lv, F. T.; Wang, S. Conjugated Polymer Nanoparticles for Light-activated Anticancer and Antibacterial Activity with Imaging Capability. Langmuir 2012, 28, 2091−2098. (32) Xing, C.; Liu, L.; Tang, H.; Feng, X.; Yang, Q.; Wang, S.; Bazan, G. C. Design Guidelines for Conjugated Polymers with Light-activated Anticancer Activity. Adv. Funct. Mater. 2011, 21, 4058−4067. (33) Storm, D. R.; Rosenthal, K. S.; Swanson, P. E. Polymyxin and Related Peptide Antibiotics. Annu. Rev. Biochem. 1977, 46, 723−763. (34) Palm, C.; Netzereab, H.; Hallbrink, M. Quantitatively Determined Uptake of Cell-penetrating Peptides in Non-mammalian Cells with An Evaluation of Degradation and Antimicrobial Effects. Peptides 2006, 27, 1710−1716. (35) Bahnsen, J. S.; Franzyk, H.; Sandberg-Schaal, A.; Nielsen, H. M. Antimicrobial and Cell-penetrating Properties of Penetratin Analogs: Effect of Sequence and Secondary Structure. Biochim. Biophys. Acta 2013, 1828, 223−232.

18520

DOI: 10.1021/acsami.7b03906 ACS Appl. Mater. Interfaces 2017, 9, 18512−18520