Selective Imaging and Inactivation of Bacteria over Mammalian Cells

Article pubs.acs.org/cm

Selective Imaging and Inactivation of Bacteria over Mammalian Cells by Imidazolium-Substituted Polythiophene Yun Huang,†,§ Harry C. Pappas,‡ Liqin Zhang,§ Shanshan Wang,§ Ren Cai,§ Weihong Tan,§ Shu Wang,∥ David G. Whitten,*,‡ and Kirk S. Schanze*,† †

Department of Chemistry, University of Texas at San Antonio, One UTSA Circle, San Antonio, Texas 78249, United States Department of Chemical and Biological Engineering, University of New Mexico, Albuquerque, New Mexico 87131, United States § Department of Chemistry, University of Florida, Gainesville, Florida 32611, United States ∥ Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡

S Supporting Information *

ABSTRACT: Antibiotic-resistant bacterial infections have become a serious public health threat. In an effort to address this threat, we develop an imidazolium-functionalized conjugated polyelectrolyte that exhibits profound light-activated biocidal activity. Here we report the synthesis, photophysical properties, and biocidal activity of a regioregular head-to-tail polythiophene substituted with cationic imidazolium units (P3HT-Im) prepared by Grignard metathesis controlled polymerization. In water, P3HT-Im has a broad absorption in the visible region and exhibited a remarkably high biocidal efficiency with both Grampositive and Gram-negative bacteria at sub-microgram per milliliter concentrations. Moreover, mammalian cell studies suggest that P3HT-Im is nontoxic to mammalian cells at concentrations of ≤20 μg/mL over a short time scale (≤1 h) in the dark and light, and the targeting rate-dependent selective mechanism is revealed. This study demonstrates the capability of P3HT-Im to achieve selective imaging and inactivation of bacteria over mammalian cells, suggesting that the polymer has significant potential for ameliorating antibiotic-resistant bacteria in a clinical setting.

■

and hydrophobic interactions.11−13 Reactive oxygen species (ROS) can be generated in the proximity of the bacterial membrane by the illumination of CPEs, causing rapid degradation of the bacterial membranes and other intracellular components.14 Recently, research on materials having selective imaging and inactivation of bacteria over mammalian cells has been of great interest.15−17 A multifunctional cationic poly(p-phenylenevinylene) derivative that enables selective inactivation of bacteria over mammalian cells at a polymer concentration of 10 μM has been reported.18 In a very recent study, branched polymeric antimicrobial peptides were reported to exhibit submicromolar

INTRODUCTION Antibiotic resistance has become a serious world health threat in the 21st century.1 Growing concerns about antibioticresistant bacteria has driven the development of new antibacterial strategies.2,3 Photodynamic inactivation of bacteria, as an alternative approach, is imbued with several favorable features, including broad-spectrum of biocidal activity and efficient inactivation of antibiotic-resistant strains, coupled with the fact that no photodynamic inactivation-resistant strains have been reported.4,5 Conjugated polyelectrolytes (CPEs) have been widely studied because they are unique materials and exhibit unique photophysical properties.3,6−9 With the inclusion of positively charged ionic side chains, such as quaternary ammoniums (R4N+) and imidazoliums,6,10,11 cationic CPEs exhibit a strong affinity for bacterial membrane surfaces through electrostatic © 2017 American Chemical Society

Received: May 2, 2017 Revised: June 27, 2017 Published: June 27, 2017 6389

DOI: 10.1021/acs.chemmater.7b01796 Chem. Mater. 2017, 29, 6389−6395

Article

Chemistry of Materials

molecular weight, and has a low polydispersity, which mitigates the effects caused by different polymer chain lengths and batchto-batch variation in properties. Because of the spin−orbit coupling introduced by the sulfur in the thiophene repeat units, P3HT-Im undergoes intersystem crossing, efficiently affording a high triplet excited state yield, which can subsequently sensitize singlet oxygen.21 Importantly, P3HT-Im can inactivate bacteria at very low concentrations (90% of the initial intensity when P3HT-Im is illuminated with blue light (λpeak ∼ 450 nm; power ∼ 1.8 mW/cm2) for 1.5 h. Given the importance of the triplet excited state in sensitizing singlet oxygen and other ROS,23 the triplet−triplet absorption of P3HT-Im was studied by nanosecond transit absorption spectroscopy. As shown in Figure 1c, P3HT-Im exhibits a long-lived transient absorption at λmax ∼ 750 nm and ground state bleaching from 350 to 480 nm. The lifetime of the transient absorption is 1.51 μs in methanol (Table 1). Given that the transient absorption of P3HT-Im has a maximal wavelength similar to that of the triplet−triplet absorption of regioregular poly(3-hexylthiophene),24 and the transient is quenched in an air-saturated solution, we presume that the P3HT-Im transient absorption is due to the triplet excited state. A triplet−triplet absorption band is also detected in H2O (Supporting Information), but it is difficult to obtain an accurate lifetime because of the weak transient absorption and comparatively long decay time (>20 μs).25 The ability of P3HT-Im to sensitize singlet oxygen is directly demonstrated by measuring singlet oxygen phosphorescence at 1270 nm in deuterated methanol (Figure 1d), and the singlet oxygen yield is determined as ΦΔ = 0.07 ± 0.01. Biocidal Studies of P3HT-Im. In vitro biocidal studies of P3HT-Im were performed with both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacteria. Following incubation with P3HT-Im at different concentrations, the bacterial cell viability was analyzed by flow cytometry (using live/dead fluorescent stains). To study the light-activated biocidal activity of P3HT-Im, S. aureus and E. coli bacterial strains were incubated separately with P3HT-Im at concentrations from 0.1 to 10 μg/mL in a physiological saline solution and then irradiated with blue-violet light (λmax ∼ 420 nm; power ∼ 2.28 ± 0.03 mW/cm2) for 1 h (dose ∼ 8.2 J/cm2). As shown in Figure 2, no significant cell death was induced by irradiation in the absence of P3HT-Im (0 μg/mL, negative control). By contrast, in the presence of 0.1 μg/mL P3HT-Im, exposure to light for 1 h killed 99.9% of S. aureus and 97.5% of E. coli. This is a biocidal agent concentration significantly lower than that used in studies with other photodynamic bacterial inactivation materials.18,26 It is clear from the striking biocidal activity that both S. aureus and E. coli bacterial cells are

Figure 2. Percentage of dead S. aureus and E. coli bacteria following incubation with P3HT-Im (0−10 μg/mL) in the dark (red and black) and irradiated with blue-violet light (λmax ∼ 420 nm; power ∼ 2.28 ± 0.03 mW/cm2, blue and green) for 1 h.

disrupted by the singlet oxygen and possibly other reactive oxygen species generated by P3HT-Im.27 The results of experiments performed in the dark at different P3HT-Im concentrations are also shown in Figure 2. In the dark, P3HT-Im exhibited pronounced biocidal activity against Gram-positive S. aureus at concentrations of ≥1 μg/mL, and at 10 μg/mL, the polymer kills 99.8% of Gram-positive S. aureus bacterial cells in 1 h. By contrast, in the dark, P3HT-Im exhibits a low killing efficiency against Gram-negative E. coli, with only ∼18% killing at a concentration of 10 μg/mL. The different killing efficiencies against Gram-positive and Gram-negative bacteria are as expected given their different cell envelope structures. Gram-positive bacteria have a thick but porous cell envelope that mainly consists of an open network of peptidoglycan and anionic teichoic and lipoteichoic acids. The negatively charged species in the bacterial cell wall provide binding sites for the cationic imidazolium side chains of P3HTIm, so that the polymer can interact with and disrupt the cell wall, which leads to the effective dark killing.18,25 In contrast, because of the protection of an extra outer lipopolysaccharide membrane,18 P3HT-Im is less likely to disrupt the cytoplasmic membrane of Gram-negative E. coli. Instead, P3HT-Im may exhibit dark toxicity against E. coli through the “ion-exchange” process,28 but the efficiency is low. In addition to the ability of P3HT-Im to sensitize singlet oxygen, and the affinity of imidazolium groups for bacterial membranes,29 we believe that two other factors contribute to the remarkable killing efficiency. First, the heteroaromatic rings increase the lipophilicity of P3HT-Im, which enhances the interaction between P3HT-Im and the bacterial cell envelope.23 Previous work from our group also supports the fact that thiophene-containing CPEs have lipophilicities that are greater 6391

DOI: 10.1021/acs.chemmater.7b01796 Chem. Mater. 2017, 29, 6389−6395

Article

Chemistry of Materials than those of poly(phenylene ethynylene)-based CPEs.30 Stronger binding allows the short-lived singlet oxygen to be generated in the proximity of the bacterial envelope and leads to efficient interaction between singlet oxygen and bacteria, possibly damaging multiple cellular components.31,32 Second, the association and insertion of P3HT-Im into the bacterial membrane leads to deaggregation of the polymer. Our previous work revealed the deaggregation process can slow nonradiative decay of the triplet state, leading to an increase in the singlet oxygen quantum yield.23 Mammalian Cell Cytotoxicity of P3HT-Im. We have also been interested in studying the interactions and killing efficiency of P3HT-Im versus bacteria in the presence of mammalian cells. As a first step, we evaluated the compatibility of P3HT-Im with mammalian cells by examining its toxicity toward HeLa cells. The Hela cell line was chosen as a practical model for the general study of cytotoxicity of P3HT-Im to mammalian cells.15,17,18,33 Mammalian cell viability analysis was performed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.34 After incubation of P3HT-Im (0−20 μg/mL) with HeLa cells at 37 °C for 24 h in the dark, the cell viability was not affected significantly (Figure 3), even at a relatively high

Figure 4. CLSM images of Gram-negative E. coli and MCF-7 cells incubated with 10 μg/mL P3HT-Im for 30 min: (a) bright field image and (b) fluorescence image. The fluorescence of P3HT-Im is colored green. The scale bar is 10 μm.

imaged by the fluorescence of P3HT-Im in a mixture with MCF-7 cells, but no fluorescence is detected inside the mammalian cells. The fluorescence signal at the periphery of the MCF-7 cells is caused by the adherence of E. coli bacteria to the MCF-7 cell surface membrane, which is observed as the result of interaction of the bacteria with the cells.35−37 In addition to the studies described above with MCF-7 cells, CLSM images of HeLa cells exposed to P3HT-Im for various incubation times provide more evidence of the weak interaction between the CPE and mammalian cells and a relatively slow rate by which the polymer penetrates the cells. As shown in Figure 5a, no P3HT-Im fluorescence is observed inside the HeLa cells after incubation for 1 h, which demonstrates that the selectivity of P3HT-Im is not specific to one kind of mammalian cell. The dynamics of penetration of P3HT-Im into HeLa cells has also been studied. Panels b and d of Figure 5 showed that there is P3HT-Im fluorescence within the HeLa cells following incubation for 8 h. It is likely that the positively charged imidazolium groups facilitate the uptake of P3HT-Im by the HeLa cells through endocytosis,38 resulting in the accumulation of the polymer within lysosomes. A targeting rate-dependent selective mechanism is proposed to achieve the selective inactivation of bacteria over mammalian cells in a short time. In a short period of time, there are significant electrostatic and hydrophobic interactions between the negatively charged phospholipids of the bacteria that promote binding of P3HTIm to bacteria, but P3HT-Im does not bind to the surface of the HeLa cells quickly because of the weaker electrostatic attraction.23 This explanation is supported the observation of less negative ζ potentials of mammalian cell membrane surfaces compared to those of bacterial membrane surfaces.18 Meanwhile, cellular endocytosis takes much longer than the inactivation of bacteria, so the different targeting rates toward bacteria and mammalian cells can be applied to achieve the selective killing of bacteria over mammalian cells.

Figure 3. Viability of HeLa cells incubated with P3HT-Im (0−10 μg/ mL) in the dark for 24 h and irradiated with blue light (λmax ∼ 450 nm; power ∼ 1.8 mW/cm2) for 1 h followed by a 24 h incubation. Cell viability assessed by the MTS assay (see Supporting Information).

concentration of P3HT-Im (20 μg/mL). To probe the toxicity of P3HT-Im in the light, cells mixed with the polymer were exposed to blue light (λmax ∼ 450 nm; power ∼ 1.8 mW/cm2) for 1 h and then incubated with the polymer in the dark for an additional 24 h prior to the MTS assay. As shown in Figure 3, the HeLa cell viability is essentially unaffected by the 1 h light exposure. It is important to note that under similar conditions of light exposure both Gram-positive and -negative bacteria are efficiently killed in separate in vitro biocidal studies (Figure 2). The viability results for the HeLa cells in the dark and light demonstrated the compatibility of P3HT-Im with mammalian cells. Targeting Rate-Dependent Selective Interaction. To further investigate the possibility of selective interaction of P3HT-Im with bacteria compared to mammalian cells, confocal laser scanning microscopy (CLSM) was utilized to detect the fluorescence from P3HT-Im after the polymer was incubated with mammalian cells for 30 min in the presence of bacteria. Figure 4 shows that E. coli can be selectively identified and

■

CONCLUSION In conclusion, the work described herein demonstrates the capability of P3HT-Im to achieve selective imaging and inactivation of bacteria in the presence of mammalian cells with a remarkably high efficiency. P3HT-Im is a metal free polymer with a simple structure, and the synthetic route has been well developed. P3HT-Im can efficiently inactivate both Gram-positive and Gram-negative bacteria with a small visible light dose (∼8.2 J/cm2) at a low concentration (0.1 μg/mL) without harming mammalian cells. The selective inactivation of 6392

DOI: 10.1021/acs.chemmater.7b01796 Chem. Mater. 2017, 29, 6389−6395

Article

Chemistry of Materials

Figure 5. CLSM images of HeLa cells incubated with 10 μg/mL P3HT-Im for 1 and 8 h: P3HT-Im fluorescent image, bright field image, fluorescent DNA label (Hoechst 33258) image, and merged image (from left to right, respectively). The fluorescence of Hoechst 33258 is colored blue. The fluorescence of P3HT-Im is colored green. Scale bars are in the bottom right in each image; for the top two rows, scale bars correspond to 50 μm, and for the bottom two rows, scale bars correspond to 10 μm. °C for 15 min). Samples were exposed to 420 nm blue light in a controlled manner using 10-lamp photoreactor (LZC-ORG, Luzchem Research Inc., Ottowa, ON). Samples were then stained with two nucleic acid stains, depending on which bacterial genus was being tested; Gram-negative E. coli cells were stained with SYTO 9 (Thermo Fisher Scientific, Waltham, MA) and propidium iodide (Thermo Fisher Scientific), while Gram-positive S. aureus cells were stained with SYTO 21 (Thermo Fisher Scientific) and propidium iodide. Samples were permitted 30 min to stain at room temperature and subsequently analyzed by flow cytometry (Accuri C6, Becton Dickinson Biosciences, San Jose, CA); 10000 events were collected per sample, at a flow rate of 11 μL/min. Two thresholds were enabled: a forward scatter threshold of 40000 and a FL1 threshold of 250. Gating schemes were defined using single-cell analysis software (FlowJo, Ashland, OR); a separate gating scheme was used for each bacterium. The fluorescence of SYTO 9 or SYTO 21 was detected with the FL1 detector; the florescence of propidium iodide was detected with the FL3 detector. All three stains are excited at 488 nm. Additionally, the FL1 detector uses a 533/30 nm filter, while the FL3 detector uses a 670 nm long pass filter. Events exhibiting high levels of green fluorescence (SYTO 9 or 21) and low levels of red fluorescence (propidium iodide) appear in quadrant 1 and were identified as being

bacteria is achieved by a strong propensity for P3HT-Im to bind to the negatively charged bacterial envelope, as well as the more rapid light-induced bacterial inactivation compared to the uptake of the polymer into mammalian cells. This study reveals the mechanism of targeting rate-dependent selectivity and the potential of P3HT-Im for addressing antibiotic-resistant bacteria in a clinical setting, especially for long-term antibiosis of traumatic wounds and burn infections, because of the good compatibility of the polymer with mammalian cells combined with the significant light-induced biocidal activity.

■

EXPERIMENTAL SECTION

Biocidal Testing. For the purpose of gauging the biocidal activity of P3HT-Im, all experiments were performed in sample volumes of 1 mL, each containing 1 × 107 cells. P3HT-Im was suspended in ultrapure water (18.2 MΩ cm at a temperature of 25 °C) at a stock concentration of 633 μg/mL. Stock concentrations exceeding 200 μg/ mL are preferable, for preventing dilution of the physiological saline solution in which the bacteria are suspended. Samples were prepared in either translucent or opaque 1.5 mL microcentrifuge tubes, which were first sterilized by autoclaving (121 6393

DOI: 10.1021/acs.chemmater.7b01796 Chem. Mater. 2017, 29, 6389−6395

Article

Chemistry of Materials ORCID

viable. Conversely, events exhibiting high levels of both green and red florescence appear in quadrant 2 and were identified as being dead, having incurred a lethal amount of membrane perturbation from P3HT-Im. The overall cell viability was determined on the basis of the following equation:

Liqin Zhang: 0000-0002-3632-4117 Weihong Tan: 0000-0002-8066-1524 Shu Wang: 0000-0001-8781-2535 Kirk S. Schanze: 0000-0003-3342-4080

viability =

Notes

no. of events in quadrant 1 (no. of events in quadrant 1) + (no. of events in quadrant 2)

The authors declare no competing financial interest.

■

To study the light-activated biocidal activity of P3HT-Im, S. aureus and E. coli bacterial strains were incubated separately with P3HT-Im at concentrations from 0.1 to 10 μg/mL in a physiological saline solution and then irradiated with blue-violet light (λmax ∼ 420 nm, power ∼ 2.28 ± 0.03 mW/cm2) for 1 h (dose ∼ 8.2 J/cm2). Cytotoxicity Analysis. The cytotoxicity of P3HT-Im was evaluated by a CellTiter 96 cell proliferation assay (Promega, Madison, WI). Briefly, cells were seeded in 96-well plates at a concentration of 4 × 104 cells per well and cultured at 37 °C for 24 h. After the cells had been washed three times with PBS, 100 μL of fresh medium containing different concentrations of P3HT-Im (1−20 μg/mL) was added. One such plate was incubated for 24 h at 37 °C in the dark. The other plate was illuminated (blue light; λpeak ∼ 450 nm; power ∼ 1.8 mW/cm2) for 1 h and then incubated for 24 h at 37 °C in the dark. CellTiter reagent (20 μL) diluted in fresh FBS free medium (100 μL) was then added to each well and incubated for 1−2 h. The absorbance (490 nm) was recorded using a microplate reader (Tecan Safire microplate reader, AG). The cell viability was determined using the absorbance of the treatment group divided by the absorbance of the medium group. Selective Staining Experiments with Bacteria and MCF-7 Cells. MCF-7 cell lines were purchased from the Cell Culture Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). MCF-7 cells were cultured in DMEM supplemented with 10% FBS and incubated in a humidified atmosphere containing 5% CO2 at 37 °C. CLSM was performed on a confocal laser scanning biological microscope (FV1000-IX81, Olympus). The cells were passaged with trypsin regularly. The P3HT-Im selective staining experiments were conducted following a reported method with some modifications.18 MCF-7 cells and E. coli bacteria were premixed in cell culture medium. Then, the P3HT-Im solution was added to make the concentration 10 μM. The suspension was incubated at 37 °C in the dark for 30 min before being scanned by the CLSM system. Cellular Uptake of P3HT-Im. The cellular uptake of P3HT-Im was studied using CLSM imaging. HeLa cells were seeded on confocal dishes and incubated at 37 °C. Twenty-four hours later, cells were washed with PBS and exposed to fresh culture medium containing either P3HT-Im for 1 or 8 h. Hoechst 33258 (3 μg/mL, Thermo Scientific) was added to the system and the mixture incubated for an additional 0.5 h to stain the nuclei. Bioimaging was performed using CLSM on a Leica TCS SP5 confocal microscope (Leica Microsystems Inc., Exton, PA) in DIC mode. An Ar laser was used for excitation of FITC.

■

ACKNOWLEDGMENTS This work was supported by funding from the Defense Threat Reduction Agency (Grant HDTRA-1-11-1-0004).

■

(1) Antimicrobial Resistance. WHO Media Centre, September 2016. (2) Maisch, T.; Szeimies, R.-M.; Jori, G.; Abels, C. Antibacterial Photodynamic Therapy in Dermatology. Photochem. Photobiol. Sci. 2004, 3, 907−917. (3) 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. (4) Jori, G.; Fabris, C.; Soncin, M.; Ferro, S.; Coppellotti, O.; Dei, D.; Fantetti, L.; Chiti, G.; Roncucci, G. Photodynamic Therapy in The Treatment of Microbial Infections: Basic Principles and Perspective Applications. Lasers Surg. Med. 2006, 38, 468−481. (5) Maisch, T. A New Strategy to Destroy Antibiotic Resistant Microorganisms: Antimicrobial Photodynamic Treatment. Mini-Rev. Med. Chem. 2009, 9, 974−983. (6) Jiang, H.; Taranekar, P.; Reynolds, J. R.; Schanze, K. S. Conjugated Polyelectrolytes: Synthesis, Photophysics, and Applications. Angew. Chem., Int. Ed. 2009, 48, 4300−4316. (7) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chemical Sensors Based on Amplifying Fluorescent Conjugated Polymers. Chem. Rev. 2007, 107, 1339−1386. (8) Bunz, U. H. F.; Seehafer, K.; Bender, M.; Porz, M. Poly(Aryleneethynylene)s (PAE) as Paradigmatic Sensor Cores. Chem. Soc. Rev. 2015, 44, 4322−4336. (9) Liu, B.; Bazan, G. C. Homogeneous Fluorescence-Based DNA Detection with Water-Soluble Conjugated Polymers. Chem. Mater. 2004, 16, 4467−4476. (10) Li, P.; Poon, Y. F.; Li, W.; Zhu, H.-Y.; Yeap, S. H.; Cao, Y.; Qi, X.; Zhou, C.; Lamrani, M.; Beuerman, R. W.; Kang, E.-T.; Mu, Y.; Li, C. M.; Chang, M. W.; Jan Leong, S. S.; Chan-Park, M. B. A Polycationic Antimicrobial and Biocompatible Hydrogel with Microbe Membrane Suctioning Ability. Nat. Mater. 2011, 10, 149−156. (11) Parthasarathy, A.; Goswami, S.; Corbitt, T. S.; Ji, E.; Dascier, D.; Whitten, D. G.; Schanze, K. S. Photophysics and Light-Activated Biocidal Activity of Visible-Light-Absorbing Conjugated Oligomers. ACS Appl. Mater. Interfaces 2013, 5, 4516−4520. (12) Yan, H.; Rengert, Z. D.; Thomas, A. W.; Rehermann, C.; Hinks, J.; Bazan, G. C. Influence of Molecular Structure on The Antimicrobial Function of Phenylenevinylene Conjugated Oligoelectrolytes. Chem. Sci. 2016, 7, 5714−5722. (13) Hinks, J.; Poh, W. H.; Chu, J. J. H.; Loo, J. S. C.; Bazan, G. C.; Hancock, L. E.; Wuertz, S. Oligopolyphenylenevinylene-Conjugated Oligoelectrolyte Membrane Insertion Molecules Selectively Disrupt Cell Envelopes of Gram-Positive Bacteria. Appl. Environ. Microbiol. 2015, 81, 1949−1958. (14) Wang, Y.; Schanze, K. S.; Chi, E. Y.; Whitten, D. G. When Worlds Collide: Interactions at the Interface between Biological Systems and Synthetic Cationic Conjugated Polyelectrolytes and Oligomers. Langmuir 2013, 29, 10635−10647. (15) Feng, G.; Mai, C.-K.; Zhan, R.; Bazan, G. C.; Liu, B. Narrow Band Gap Conjugated Polyelectrolytes for Photothermal Killing of Bacteria. J. Mater. Chem. B 2015, 3, 7340−7346. (16) Li, K.; Lei, W.; Jiang, G.; Hou, Y.; Zhang, B.; Zhou, Q.; Wang, X. Selective Photodynamic Inactivation of Bacterial Cells over

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b01796. Materials, instrumentation and methods, Scheme S1, synthetic route, Figures S1−S10, and additional references (PDF)

■

REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. 6394

DOI: 10.1021/acs.chemmater.7b01796 Chem. Mater. 2017, 29, 6389−6395

Article

Chemistry of Materials Mammalian Cells by New Triarylmethanes. Langmuir 2014, 30, 14573−14580. (17) Zhao, Q.; Li, J.; Zhang, X.; Li, Z.; Tang, Y. Cationic Oligo(thiophene ethynylene) with Broad-Spectrum and High Antibacterial Efficiency under White Light and Specific Biocidal Activity against S. aureus in Dark. ACS Appl. Mater. Interfaces 2016, 8, 1019−1024. (18) Zhu, C.; Yang, Q.; Liu, L.; Lv, F.; Li, S.; Yang, G.; Wang, S. Multifunctional Cationic Poly(p-phenylene vinylene) Polyelectrolytes for Selective Recognition, Imaging, and Killing of Bacteria over Mammalian Cells. Adv. Mater. 2011, 23, 4805−4810. (19) Lam, S. J.; O’Brien-Simpson, N. M.; Pantarat, N.; Sulistio, A.; Wong, E. H. H.; Chen, Y.-Y.; Lenzo, J. C.; Holden, J. A.; Blencowe, A.; Reynolds, E. C.; Qiao, G. G. Combating Multidrug-Resistant GramNegative Bacteria with Structurally Nanoengineered Antimicrobial Peptide Polymers. Nat. Microbiol. 2016, 1, 16162. (20) Osaka, I.; Mccullough, R. D. Advances in Molecular Design and Synthesis of Regioregular Polythiophenes. Acc. Chem. Res. 2008, 41, 1202−1214. (21) Burrows, H. D.; Seixas De Melo, J.; Serpa, C.; Arnaut, L. G.; Miguel, M. D. G.; Monkman, A. P.; Hamblett, I.; Navaratnam, S. Triplet State Dynamics on Isolated Conjugated Polymer Chains. Chem. Phys. 2002, 285, 3−11. (22) Zhai, L.; Pilston, R. L.; Zaiger, K. L.; Stokes, K. K.; Mccullough, R. D. A Simple Method to Generate Side-Chain Derivatives of Regioregular Polythiophene via the GRIM Metathesis and PostPolymerization Functionalization. Macromolecules 2003, 36, 61−64. (23) Corbitt, T. S.; Ding, L.; 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. (24) Kraabel, B.; Moses, D.; Heeger, A. J. Direct Observation of the Intersystem Crossing in Poly(3-octylthiophene). J. Chem. Phys. 1995, 103, 5102−5108. (25) Parthasarathy, A.; Pappas, H. C.; Hill, E. H.; Huang, Y.; Whitten, D. G.; Schanze, K. S. Conjugated Polyelectrolytes with Imidazolium Solubilizing Groups. Properties and Application to Photodynamic Inactivation of Bacteria. ACS Appl. Mater. Interfaces 2015, 7, 28027− 28034. (26) Strassert, C. A.; Otter, M.; Albuquerque, R. Q.; Hoene, A.; Vida, Y.; Maier, B.; De Cola, L. Photoactive Hybrid Nanomaterial for Targeting, Labeling, and Killing Antibiotic-Resistant Bacteria. Angew. Chem., Int. Ed. 2009, 48, 7928−7931. (27) Chambon, S.; Rivaton, A.; Gardette, J.-L.; Firon, M. Reactive Intermediates in the Initiation Step of the Photo-Oxidation of MDMO-PPV. J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 6044− 6052. (28) Wang, Y.; Corbitt, T. S.; Jett, S. D.; Tang, Y.; Schanze, K. S.; Chi, E. Y.; Whitten, D. G. Direct Visualization of Bactericidal Action of Cationic Conjugated Polyelectrolytes and Oligomers. Langmuir 2012, 28, 65−70. (29) Hatz, S.; Poulsen, L.; Ogilby, P. R. Time-Resolved Singlet Oxygen Phosphorescence Measurements from Photosensitized Experiments in Single Cells: Effects of Oxygen Diffusion and Oxygen Concentration. Photochem. Photobiol. 2008, 84, 1284−1290. (30) Ding, L.; Chi, E. Y.; Schanze, K. S.; Lopez, G. P.; Whitten, D. G. Insight into the Mechanism of Antimicrobial Conjugated Polyelectrolytes: Lipid Headgroup Charge and Membrane Fluidity Effects. Langmuir 2010, 26, 5544−5550. (31) 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. (32) Dahl, T. A.; Midden, W. R.; Hartman, P. E. Comparison of Killing of Gram-Negative and Gram-Positive Bacteria by Pure Singlet Oxygen. J. Bacteriol. 1989, 171, 2188−2194. (33) Li, Z.; Yang, R.; Yu, M.; Bai, F.; Li, C.; Wang, Z. L. Cellular Level Biocompatibility and Biosafety of ZnO Nanowires. J. Phys. Chem. C 2008, 112, 20114−20117.

(34) Zhang, L.; Zhu, G.; Mei, L.; Wu, C.; Qiu, L.; Cui, C.; Liu, Y.; Teng, I. T.; Tan, W. Self-Assembled DNA Immunonanoflowers as Multivalent CpG Nanoagents. ACS Appl. Mater. Interfaces 2015, 7, 24069−24074. (35) Pizarro-Cerda, J.; Cossart, P. Bacterial Adhesion and Entry into Host Cells. Cell 2006, 124, 715−727. (36) Rottner, K.; Stradal, T. E. B.; Wehland, J. Bacteria-Host-Cell Interactions at the Plasma Membrane: Stories on Actin Cytoskeleton Subversion. Dev. Cell 2005, 9, 3−17. (37) Krachler, A. M.; Ham, H.; Orth, K. Outer Membrane Adhesion Factor Multivalent Adhesion Molecule 7 Initiates Host Cell Binding during Infection by Gram-Negative Pathogens. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 11614−11619. (38) Li, L.; Raghupathi, K.; Yuan, C.; Thayumanavan, S. Surface Charge Generation in Nanogels for Activated Cellular Uptake at Tumor-Relevant pH. Chem. Sci. 2013, 4, 3654−3660.

6395

DOI: 10.1021/acs.chemmater.7b01796 Chem. Mater. 2017, 29, 6389−6395