Synthesis of Multifunctional Cationic Poly (p-phenylenevinylene) for

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Synthesis of multifunctional cationic poly(p-phenylene vinylene) for selectively killing bacteria and lysosome-specific imaging Zhuo Chen, Huanxiang Yuan, and Haiyan Liang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01609 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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ACS Applied Materials & Interfaces

Synthesis of multifunctional cationic poly(p-phenylene vinylene) for selectively killing bacteria and lysosome-specific imaging Zhuo Chen, Huanxiang Yuan*, Haiyan Liang Department of Chemistry, School of Science, Beijing Technology and Business University, Beijing 100048, P. R. China. ABSTRACT In this work, a cationic polymer was synthesized to bear quaternized N-methyl-imidazole groups in the side chains. Positively charged PPV-M could selectively bind to Gram-negative and Gram-positive bacteria over fungi and exhibit enhanced antibacterial activity with the aid of white light because PPV-M could sensitize oxygen to generate reactive oxygen species (ROS) that would damage bacteria. In addition, green fluorescent and positively charged PPV-M has ability to enter mammalian cells and be specifically accumulated in lysosome. Moreover, PPV-M could stay in live cells for a relatively long time, which implies that PPV-M is potential to be a long-term imaging agent. KEYWORDS:

multifunctional

cationic

polymer,

reactive

oxygen

species,

photosensitizer, selective antibacterial activity, lysosome-specific imaging Bacterial infection leads to nearly one-third of global deaths each year.1 Antibiotics are the most widely used to treat bacterial illnesses.2 But the misuse/overuse of antibiotics results in the generation of antibiotic-resistant bacteria, which would seriously threaten the global public health.3-7 With the rapid development of antibiotic resistance of bacteria, it is urgent to explore new materials

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or systems to fight with bacterial infections. Photodynamic therapy (PDT), as a photoinduced therapy, has been successfully applied to treat bacterial infections in clinic.8-9 Resistant bacteria could be easily destroyed via PDT without rapid development of resistance.10-11 Photosensitizer is one of the most important component in PDT to provide the production of reactive oxygen species (ROS) which are toxic toward cells and microbes.12 Recently, quaternary ammonium based conjugated polymers were synthesized to be utilized as photosensitizers for antimicrobial application because they have potential capability to sensitize oxygen molecules to generate ROS with white light irradiation.13-17 Conjugated polymers bearing quaternary ammonium group which simultaneously possess excellent biocompatibility, outstanding photosensitivity and imaging ability are desired to be used in PDT. Despite the great efforts in application of cationic conjugated polymers for killing pathogens, few conjugated polymers are synthesized to selectively bind and kill bacteria over fungi. Here, we synthesized a cationic polymer (PPV-M) bearing quaternized N-methyl-imidazole groups in the side chains. As illustrated in Scheme 1a, PPV-M could selectively bind to Gram-positive (G+) and Gram-negative (G-) bacteria rather than fungi. Upon the irradiation of white light, bacteria treated with PPV-M could be completely damaged. However, PPV-M could not kill fungi owing to the non-interaction between PPV-M and fungi. Additionally, PPV-M has excellent biocompatibility and could be successfully used as lysosome-specific imaging agent.


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Scheme 1 (a) Schematic illustration of antimicrobial process of PPV-M. (b) Synthesis of cationic PPV-M. The synthetic route of PPV-M is illustrated in Scheme 1b. Reaction of PPV-Br with N-methyl-imidazole afforded cationic PPV-M in a 53% yield. Because of the quaternized N-methyl-imidazole groups, the positively charged PPV-M is water-soluble. The photophysical properties of PPV-M were characterized in water. As shown in Figure S1a, PPV-M exhibits a maximum adsorption at 465 nm and the maximum emission wavelength is 554 nm with an absolute fluorescence quantum yield of 1.8%.



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The generation of reactive oxygen species (ROS) sensitized by PPV-M was firstly investigated. 2, 7-dichlorofluorescein diacetate (DCFH-DA) was applied as a probe of ROS, which could be hydrolyzed to 2, 7-dichlorofluorescein (DCFH) treated by NaOH aqueous solution followed by converting to 2, 7-dichlorofluorescein (DCF) with high fluorescence quantum in the presence of ROS.18 As can be seen in Figure S1b, the fluorescence of DCF significantly increased in the presence of PPV-M under the irradiation of white light at a fluence rate of 5 mW⋅cm-2 and the control group in the absence of PPV-M displays no obvious change with excitation of 488 nm. The results implies that PPV-M has strong capability to sensitize the surrounding oxygen to produce ROS which can easily damage microbes.

Figure 1. CLSM images of E. coli, S. aureus and C. albicans treated with 10 µM, 30 µM and 60 µM PPV-M, respectively. To directly verify whether PPV-M could bind to negatively charged microbes,


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confocal laser scanning microscopy (CLSM) was utilized to study the interaction between cationic PPV-M and microbes. Ampr E. coli, S. aureus and C. albicans were selected to represent Gram-negative bacteria, Gram-positive bacteria and fungi respectively. Figure 1 displays that green fluorescence of E. coli and S. aureus treated with PPV-M was detected and the fluorescence intensity of E. coli and S. aureus increased with the increase of PPV-M concentration, which indicates that PPV-M could successfully adsorb onto the bacteria and provides the possibility to kill them; while few C. albicans treated with PPV-M were stained demonstrating that PPV-M cannot interact with C. albicans. Zeta potential characterization further provides more insights into the interaction of PPV-M with negatively charged microbes. As exhibited in Figure 2a, the zeta potential of untreated E. coli is -52.9 mV while it changes to -38.8 mV when treated with 10 µM of PPV-M, and the zeta potential of E. coli increases to -9.3 mV when 60 µM of PPV-M is added to E. coli. As for the case of S.

aureus, the zeta potential was to grow from -16.0 to 4.6 after treated with 60 µM of PPV-M. The zeta potentials of E. coli and S. aureus incubated with PPV-M became more positive than E. coli and S. aureus alone, which demonstrates that PPV-M binds to the surface of Gram-negative and Gram-positive bacteria. Nevertheless, the potentials of C. albicans almost keep unchanged with treatment under different concentrations of PPV-M, indicative of the non-interaction between PPV-M and C.

albicans. The potential data are consistent with the CLSM imaging results. Thus, PPV-M selectively binds to Gram-negative and Gram-positive bacteria rather than fungi. Universally, both Gram-negative bacteria and Gram-positive bacteria have a



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typical porous and interconnected peptidoglycan layer as the primary component of cell wall and abundant negative charges provided by lipopolysaccharide or teichoic acids,19 which could facilitate the binding and inserting of PPV-M toward these two species.11 For C. albicans, relatively less negative charges and dense cell wall mainly composed of mannatide, glucans and chitin lead to the lack of coating of PPV-M on the surface.20 Therefore, the different binding ability of PPV-M toward microorganisms is dependent on their diverse envelope structure characteristics.

Figure 2. (a) Zeta potentials of E. coli, S. aureus and C. albicans before and after treated with 10 µM, 30 µM and 60 µM PPV-M, respectively. (b) Antibacterial activity of PPV-M toward E. coli in the absence and presence of white light irradiation. (c)


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Antibacterial activity of PPV-M toward S. aureus in the absence and presence of white light irradiation. After confirming the ROS generation sensitized by PPV-M and the specific interaction of PPV-M with E. coli and S. aureus, the selective antibacterial activity of PPV-M was evaluated using a standard colony counting method. Antibacterial experiments were conducted under white light irradiation and in dark. As shown in Figure 2b, the killing efficiency is from 46% to 73% in the PPV-M concentration range of 10 µM~60 µM without white light irradiation. Because of the cationic quaternized N-methyl-imidazole groups which have affinity toward negatively charged bacteria followed by damaging the membrane of bacteria, PPV-M itself could kill a proportion of Ampr E. coli. For the case of white light irradiation, PPV-M kills more than 95% E. coli in a relatively low concentration of 10 µM and kills more than 99% E. coli when the concentration of PPV-M are 30 µM and 60 µM. This significant increase of antibacterial capacity of PPV-M attributes to the ROS generation sensitized by PPV-M upon the irradiation of white light. As for Gram-positive S.

aureus, colony counting shows that the killing efficiency is approximately 30% and it reaches more than 99% in a relatively high PPV-M concentration of 60 µM in the absence of white light due to the quaternized N-methyl-imidazole groups. Moreover, upon the irradiation of white light, PPV-M in a low concentration of 10 µM exhibited more than 99% killing efficiency implying the serious damage of Gram-positive S.

aureus. (Figure 2c) It is noted that E. coli and S. aureus untreated with PPV-M cannot be affected under white light irradiation condition (75 mW⋅cm-2 for 7 min) used in the



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antibacterial experiments. To acquire more evidence about the selective antibacterial activity of PPV-M, antifungal experiment against C. albicans was performed. According to the colony counting (Figure S4), PPV-M could not affect the viability of

C. albicans, which demonstrates the selectively killing ability of PPV-M toward Gram-negative and Gram-positive bacteria.

Figure 3. (a) Morphology of E. coli incubated with 10 µM, 30 µM and 60 µM PPV-M in dark and upon white light irradiation. The scale bar is 1 µm. (b) CLSM images of MCF-7 cells incubated with 20 µM PPV-M co-stained with 1 µM LysoTracker. The excitation wavelengths of PPV-M and LysoTracker are 488 nm and 559 nm respectively. To further explore the mechanism of antibacterial activity of PPV-M, scanning electron microscope (SEM) was applied to observe the surface changes of E. coli, S.

aureus and C. albicans before and after incubation with PPV-M. As displayed in


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Figure 3a, the control groups including E. coli without PPV-M and without white light irradiation exhibit clear edges and intact morphology, which is consistent with the antibacterial experiments and further confirms that white light condition used here has no effects on the bacteria. With treatment of PPV-M alone, sunken and collapsed surfaces of E. coli were visualized, which indicates the damage of bacterial membrane. Upon the irradiation of white light, E. coli incubated with PPV-M exhibit severely split and fused morphology. White light illumination causes greater damage toward bacteria. For S. aureus, merged surfaces were found in the group treated with high concentration of PPV-M in the absence and presence of light irradiation. (Figure S5) SEM characterization produces results essentially in agreement with the antibacterial experiments. As for C. albicans, no obvious change of the morphology was observed before and after treatment with different concentrations of PPV-M with or without white light irradiation. (Figure S6) The direct results from SEM characterization are in agreement with the zeta potential and antimicrobial experiments.

Excellent biocompatibility toward mammalian cells is of great importance for a potential antibacterial agent. Therefore, cell viability analysis of PPV-M to MCF-7 cells was performed using a standard MTT method. Figure S7 shows that MCF-7 cells remain more than 90% viability in the range of antibacterial concentrations, which implies the low cytotoxicity of PPV-M. On the basis of the fluorescence property and excellent biocompatibility of PPV-M, we subsequently investigated the interaction of PPV-M with MCF-7 cells.



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After incubated with PPV-M for 8 h, MCF-7 cells were monitored using CLSM. CLSM images show that PPV-M entered cells and distributed in cytoplasm. In order to further confirm the location of PPV-M in cells, LysoTracker, an organelle dye for specifically identifying lysosome, was applied to analyze the colocalization. As shown in Figure 3b, the green fluorescence of PPV-M and the red fluorescence of LysoTracker

overlay

very

well,

demonstrating

a

characteristic

lysosome

colocalization. Moreover, the Pearson’s correlation coefficient calculated by the software based on the merged image of PPV-M and LysoTracker channels is 0.92, which quantifies that PPV-M mainly accumulates in lysosome.21 These results suggest that PPV-M could be utilized as potential lysosome-specific imaging agent. Additionally, the imaging ability of PPV-M encourages us to explore whether it can retain for a long time in cells to be potential long-term imaging materials. Compared with commercial LysoTracker which is extruded by cells after the third day, PPV-M could remain in cells for 7 days at least. (Figure S8) Thus, PPV-M is very promising to be a long-term imaging agent.

In summary, we have synthesized a multifunctional cationic polymer bearing quaternized N-methyl-imidazole groups in the side chains for selectively and effectively killing bacteria and specifically imaging of lysosome. The positively charges endow PPV-M with binding ability to negatively charged bacteria and mammalian cells. Zeta potential measurement confirm the selective interaction


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between PPV-M and bacteria. PPV-M could sensitize oxygen to generate cytotoxic ROS under white light irradiation, which affords PPV-M the potential to kill bacteria in light irradiation condition. Antibacterial experiments demonstrate that PPV-M itself could kill a proportion of bacteria but white light significantly improve the killing efficiency, which indicates that PPV-M has efficient light toxicity. In addition, because of the green fluorescence and positive charges of PPV-M, it could be successfully used as cell imaging agent and surprisingly PPV-M was highly accumulated in lysosome of cells, which implies that PPV-M could be a potential lysosome-specific imaging material. Moreover, PPV-M has good retention capacity in cells to be a promising long-term imaging agent. Therefore, this new synthesized multifunctional cationic conjugated polymer combines selective photo-excited bacteria killing ability and lysosome-specific imaging property, which is a simple system to achieve collaborative biological applications.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. Experimental details, spectra, plate counting, SEM, cell viability, CLSM and photostability results.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] (Huanxiang Yuan)



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ORCID Huanxiang Yuan: 0000-0001-5344-3655

ACKNOWLEDGE We are grateful to the National Natural Science Foundation of China (No. 21504002,) and Beijing Technology and Business University fund project (No. LKJJ2016-20).

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Synthesis of Multifunctional Cationic Poly (p-phenylenevinylene) for

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