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Biofilm Inhibition and Elimination Regulated by Cationic Conjugated Polymers Pengbo Zhang, Shengliang Li, Hui Chen, Xiaoyu Wang, Libing Liu, Fengting Lv, and Shu Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 08 May 2017 Downloaded from http://pubs.acs.org on May 9, 2017

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ACS Applied Materials & Interfaces

Biofilm Inhibition and Elimination Regulated by Cationic Conjugated Polymers Pengbo Zhang, Shengliang Li, Hui Chen, Xiaoyu Wang, Libing Liu*, Fengting Lv and Shu Wang* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China

KEYWORDS: conjugated polymer, biofilm, inhibition, elimination, ROS

ABSTRACT: In this work, we demonstrate that water-soluble conjugated polymers (PFP) have the ability to inhibit biofilm formation and eradicate mature established biofilm using reactive oxygen species (ROS) produced by PFP under white light irradiation. Upon addition of PFP to planktonic Staphylococcus aureus (S. aureus), electrostatic interactions bring cationic PFP to the surface of S. aureus which possess negative charges. As the amount of PFP coated on S. aureus become saturated, the interactions of bacteria to bacteria and bacteria to surface may be disrupted, resulted in reduced biofilm formation. While after biofilm mature, those PFP on the surface of biofilm can generate ROS under white light irradiation which has the ability to inactivate bacteria nearby. Once the biofilm broken, PFP can penetrate through biofilm and continuously generate ROS under irradiation, resulted in biofilm disruption. As a consequence,

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this makes conjugated polymers a very promising material for the disruption of biofilm in biomedical and industrial applications. INTRODUCTION An increasing number of bacterial infections have represented a major problem in human health care, resulting in invasive infections that are related to high mortality.1 However, almost 80% of all bacterial infections occurring in the human body are biofilm-associated.2 When growing on surface, many bacteria can form complex multicellular communities, known as biofilm, in which bacteria are held together and embedded in a self-produced extracellular polymeric substances (EPS) mainly composed of polysaccharides, proteins, lipids and extracellular DNA.3-5 The established biofilms can induce a great number of persistent infections including chronic lung, wound and medical devices infections.6,7 Bacteria within biofilms possess different cell wall composition, growth rate, EPS components and even gene type. In comparison to planktonic bacteria, the inhibited penetration of the drugs, limited nutrition supply and slow growth rate cause bacteria within biofilms become less susceptibility to antibiotics.7,8 This may lead to the formation of multi-antibiotic resistant (MAR) bacteria which cannot be killed by many kinds of drugs. As the physical and chemical structure of biofilm matrix is critical for the persistent resistance to antimicrobial agents, many studies have concentrate on biofilm inhibition through the activation of intrinsic bacterial responses. For example, Boles and co-workers find that activation of the agr quorum sensing system of established Staphylococcus aureus (S. aureus) biofilm is crucial for biofilm reduction through increasing the level of extracellular protease needed for detachment.9 An alternate way for biofilm disruption is the use of reagents which can degrade the matrix and finally eliminate biofilm, such as surfactant-like molecules,10,11 proteases,12 DNase13 and other enzymes targeting biofilm matrix components14.


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Recently, D-amino acids isolated from the supernatants of Bacillus subtilis biofilm medium are shown to inhibit biofilm formation in fresh cultures.15 Although much work in the inhibition and elimination of biofilm have been reported, the development of more efficient and simple methods that do not induce bacterial resistance is still a research hotspot. Due to the unique electronic and optical properties, water-soluble conjugated polymers (CPs) have attracted much attention in their biological applications.16,17 Compared with small molecule counterparts, repeated light absorbing units in CPs allow the excitation energy transfer along the whole backbone to the acceptor, resulting in amplified fluorescence signals of the acceptor which greatly improves the detection sensitivity.18 With these outstanding properties, CPs have been successfully used for biomacromolecules detection, fluorescence imaging, photo-assisted protein inactivity and bacteria detection.19-29 Recently, Whitten et al. demonstrated that cationic poly(pphenyleneethyneylenes)s (PPEs) with quaternary ammonium (QA) groups as side chain can bind to the surface of bacteria and exhibit effective antibacterial capability against a variety of bacteria.30-32 The current state of the art in the field focuses on their applications in highly sensitive sensing and imaging, however, few studies have been demonstrated to utilize CPs in biofilm disruption and elimination. It has been reported that positive charges on the cationic CPs provide tight electrostatic interactions toward negatively charged sites at the membrane surface of gram-positive and gram-negative bacteria.28 In our previous work, we have shown that cationic CPs can induce bacteria quorum sensing (QS) system and prolong the time duration of QS signal molecules production which is critical for biofilm formation.33 In addition, under irradiation of proper light, many CPs can be excited and react with molecular oxygen in the surrounding to generate reactive oxygen species (ROS) with efficient antibacterial activity. Taking advantages of these properties, we demonstrate that cationic conjugated polymer (CCP)


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can prevent biofilm formation without impairing bacterial viability and eradicate mature established biofilm using ROS produced by CCP under irradiation of white light.

Scheme 1. The inhibition of biofilm formation and elimination of mature established biofilm using PFP. RESULTS AND DISCUSSION In this study, biofilm inhibition and elimination are regulated by CCP. S. aureus (ATCC 6538) is chosen as the bacteria to form biofilms. S. aureus is a species of gram-positive bacteria which exhibits

negative

charges

on

the

membrane

surface.34

Poly{[(9,9-bis(6’-N,N,N-

trimethylammonium)hexyl) fluorenylene phenylene]dibromide} (PFP) with positively charged QA groups on the exterior is used as model conjugated polymer.27,35 PFP was synthesized according to the procedure in the literature.36 The biocidal activity of PFP relies on the insertion of QA into the cell membrane and the ability to generate ROS by sensitizing oxygen molecules around.17,31,37 The inhibition of biofilm formation and elimination of mature established biofilm


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using PFP are demonstrated in Scheme 1. Upon adding PFP to the planktonic S. aureus, cationic PFP can bind to the negatively charged sites at the surface of S. aureus through electrostatic interactions. After coating with PFP, S. aureus become cationic and the interactions of bacteria to bacteria and bacteria to surface may be disrupted, resulted in reduced biofilm formation. While adding PFP to the mature S. aureus biofilm, electrostatic interactions bring them to the surface of biofilm. Under irradiation of white light, PFP sensitizes oxygen molecules in the surroundings to generate ROS which has the ability to inactivate bacteria nearby. As biofilm broken, PFP can penetrate through biofilm and continuously generate ROS under irradiation, resulted in biofilm disruption. In order to investigate whether PFP is a potent inhibitor of biofilm formation, biofilm measured by crystal violet staining is detected. We firstly proved that PFP itself did not affect the viability of S. aureus according to the data of growth curve (Figure S1). Then different concentrations of PFP were added into planktonic S. aureus. After incubation, the number of bacteria in biofilm was quantified by crystal violet stain and traditional surface plating method. The photos of biofilm stained with crystal violet were shown in Figure 1a, and quantification of biofilm by absorbance of crystal violet at 590 nm was shown in Figure 1b. The colony-forming units (CFU) of S. aureus biofilm after addition of different concentrations of PFP was shown in Figure S2. The results confirmed that detectable biofilm was formed when incubation without PFP and no obvious changes were observed after adding 1-20 µM PFP. While upon addition of 50 µM and 100 µM PFP, biofilm was formed with less intense crystal violet staining, indicative of successful inhibition of biofilm formation. To afford more insights into the mechanism of the inhibitory effect, zeta (ζ) potential was performed to investigate the interactions between S. aureus and PFP. Due to the components of


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Figure 1. (a) Biofilm stained with crystal violet. (b) Quantification of biofilm by absorbance of crystal violet. *** P < 0.001 with respect to blank (Tukey's test). (c) ζ potentials of S. aureus incubated with different concentrations of PFP. (d) CLSM image of S. aureus incubated with 50 µM PFP. The fluorescence of PFP is highlighted in blue. bacteria surface (mainly lipids and proteins), S. aureus alone possess considerably negative charges. Upon addition of PFP, ζ potentials of S. aureus became cationic, indicating the successful binding of PFP to the outer membranes of S. aureus through electrostatic interactions. As shown in Figure 1c, the surface charges of S. aureus turned to be more positive with increased concentration of PFP. When the concentration reached 50 µM, ζ potential was high enough and remained almost unchanged at concentrations higher than 50 µM, which meant the concentration of PFP was high enough to wrap all S. aureus in. Confocal laser scanning


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microscopy (CLSM) was utilized to further explain the phenomenon. As shown in Figure 1d and S3, S. aureus and the blue fluorescence of PFP-G2 were clearly overlapped and all bacteria were shown blue colour. By coating S. aureus with cationic PFP, the interactions of bacteria to bacteria and bacteria to surface were limited, resulted in inhibited biofilm formation. Meanwhile, PFP and S. aureus cells were aggregated to agglomerates while control experiments showed that S. aureus without PFP did not aggregate (Figure S3), indeed supporting the interactions between S. aureus and PFP. Extracellular polysaccharide induced aggregation in the biofilm formed by S. aureus was stained by fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) which selectively bind to the mannose and glucose residues presented in biofilm matrix.38 As shown in Figure S4, green fluorescence of FITC-ConA was reduced after addition of 50 µM and 100 µM PFP, resulted in reduced polysaccharides formation. These results demonstrate that PFP has the ability to inhibit biofilm formation by coating S. aureus through electrostatic interactions. To verify the generation of ROS from PFP under white light irradiation, 2’, 7’dichlorofluorescein diacetate (DCFH-DA), a ROS sensitive probe, was utilized. In the presence of ROS, DCFH-DA was converted to non-emissive 2’, 7’-dichlorofluorescin (DCFH) and finally transformed into highly fluorescent 2’, 7’-dichlorofluorescein (DCF).39 Compared with conventional photosensitizers, PFP generate 10 times more ROS than porphyrins (Figure S5). As shown in Figure 2a, upon irradiating DCFH in the presence of different concentrations of PFP under white light, concentration-dependent increase of fluorescence intensity at 525 nm (characteristic emission of DCF) was detected, while control experiment without PFP is basically maintained at the baseline level. It is noted that PFP itself cannot be excited at 488 nm which is used to excite DCF, so these results confirm the considerable generation of ROS originated from PFP. Motivated by the excellent ROS generation ability of PFP, together with strong electrostatic


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interactions between PFP and S. aureus, photodynamic anti-biofilm ability of PFP has been developed. PFP was firstly added to the mature biofilm. After the biofilm coated with PFP, white light of different time duration were introduced to initiate photodynamic biofilm detachment

Figure 2. (a) ROS sensitized by different concentrations of PFP under white light irradiation detected by DCFH. [DCFH] = 40 µM. (b) Biofilm detachment activity in the presence and absence of PFP toward S. aureus under white light irradiation of different time duration. [PFP] =


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20 µM. (c) CFU of S. aureus biofilm under white light irradiation after treatment in the presence and absence of PFP. *P < 0.05, **P < 0.05, and ***P < 0.001 vs PFP under different time duration of white light irradiation or in the indicated pare by Tukey's test. without affecting the temperature of sample solution (Figure S6). The results were shown in Figure 2b and 2c by a traditional surface plating method. When the irradiation time increased from 5 min to 15 min, the inhibition ratio of PFP increased sharply and the inhibition ratio of blank was still less than 10%, demonstrating that illumination itself did not affect the viability of biofilm. When the irradiation time further increased from 15 min to 25 min, the inhibition ratio of PFP increased slowly along with a noticeable enhancement of blank which could not be neglected. The inhibition ratio of PFP to blank reached the highest value at 15 min. These results suggest that ROS produced by PFP is a main factor accounts for the elimination of biofilm. To better understand the effect of photodynamic anti-biofilm ability of PFP on the viability of bacteria within the biofilms, a BacLight Live/Dead viability kit was utilized to stain bacteria for CLSM. The kit contains two fluorescent dyes: SYTO9 penetrates both live and dead bacteria while propidium iodide (PI) penetrates bacteria with damaged membranes and quenches the fluorescence of SYTO9.40 As a consequence, live bacteria with intact membranes can be stained by SYTO9 and fluoresce green, while dead bacteria with damaged membranes can be stained by PI and fluoresce red. In biofilms incubated without PFP, CLSM images showed that majority of the bacteria show green fluorescence, demonstrating that illumination for 15 min did not affect the viability of biofilm (Figure 3a). The images of biofilm incubated with PFP in the absence of white light were similar to those without PFP. While upon irradiation for 15 min after PFP treatment, bacteria in biofilms was stained with red and green. When further extending


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irradiation time to 25 min, bacteria which mostly fluoresce red were observed and biofilms appeared to be less dense compared to the controls. The results verified that ROS generated by PFP can kill the bacteria within biofilm. Scanning electron microscope (SEM) was then

Figure 3. (a) CLSM images of S. aureus biofilms stained by PI & SYTO9. (b) SEM images of S. aureus biofilms. Biofilms were grown for 24 h and treated with 20 µM PFP, under white light


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irradiation of 0 min, 5 min, 15 min and 25 min. The scale bar is 20 µm and the original magnification is ×2000. employed to get more insights into the morphological changes of biofilms (Figure 3b). Large bacterial aggregates coated by EPS were observed for control experiments (biofilm without PFP or/and without white light irradiation). Under irradiation for 15 min in the presence of PFP, significant removal of the biofilms and smaller size of aggregates were observed. Further extending irradiation time to 25 min caused intense reduction of cell density and dispersion of the biofilms, indicating the loss of bacteria within the biofilms. The reason may due to those bacteria within biofilms dead with damaged membranes by ROS generated from PFP which caused reduced bacteria to bacteria interaction and bacteria to matrix binding force, resulted in disruption and detachment in biofilms. These direct observations from SEM images are consistent with the CLSM images and biofilm detachment experiments. CONCLUSION In conclusion, a multifunctional conjugated polymer with positive charges on the side chains can be used for the inhibition of bacteria biofilm formation and elimination of mature established biofilms. The method takes advantages of the unique electronic and optical properties and the singlet oxygen generation ability of conjugated polymers. On one hand, the positive charges of PFP guarantee its adsorption to the negatively charged membrane of S. aureus through electrostatic interactions. ζ potentials of S. aureus become more positive with increased concentration of PFP and the amount of PFP bound to S. aureus reaches saturation when the concentration of PFP increased to 50 µM. By wrapping all S. aureus in, PFP has the ability to disrupt interactions of bacteria to bacteria and bacteria to surface. On the other hand, PFP can


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sensitize oxygen molecules around to generate ROS which has the ability to inactivate bacteria nearby under irradiation of white light. After PFP binding to the surface of mature biofilm, ROS generated by PFP can kill the bacteria within biofilm. Once biofilm broken, PFP can penetrate through biofilm and continuously generate ROS under irradiation, resulted in biofilm disruption. As a consequence, conjugated polymers show great potential for the disruption of biofilm in treating biofilm-associated infections which are accessible to light.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Detailed experimental procedures including the growth curve of S. aureus, biofilm stained by crystal violet, ζ potential measurements, CLSM characterization of S. aureus, biofilm stained by FITC-ConA, ROS measurements, biofilm detachment experiments, CLSM characterization of S. aureus biofilm, SEM characterization and additional Figures S1−S6.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Notes


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The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors are grateful to the National Natural Science Foundation of China (Nos. 21473220), the Major Research Plan of China (No. 2013CB932800) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB12030300).

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Biofilm Inhibition and Elimination Regulated by ... - ACS Publications

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