pH-Sensitive Theranostic Nanoparticles for Targeting Bacteria with

Article Cite This: ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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pH-Sensitive Theranostic Nanoparticles for Targeting Bacteria with Fluorescence Imaging and Dual-Modal Antimicrobial Therapy Peng Liu,† Li Qun Xu,‡ Gang Xu,† Dicky Pranantyo,† Koon-Gee Neoh,† and En-Tang Kang*,† †

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Kent Ridge 117585, Singapore ‡ Institute for Clean Energy and Advanced Materials, Faculty of Materials and Energy, Southwest University, Chongqing 400715, P. R. China

ACS Appl. Nano Mater. Downloaded from pubs.acs.org by 79.110.17.112 on 10/31/18. For personal use only.

S Supporting Information *

ABSTRACT: Bacterial infections with severely damaging consequences are serious threats to the public healthcare system. A pHsensitive theranostic system was fabricated for targeting bacteria with fluorescence imaging and dual-modal antimicrobial therapy (cationic and photodynamic therapies). Poly(poly(ethylene glycol) methyl ether methacrylate)-b-poly(2-(diisopropylamino) ethyl methacrylate-co-2-hydroxyethyl methacrylate)-chlorin e6 (PPEGMA-b-P(DPA-co-HEMA)-Ce6) was synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerization and post-modification. The self-assembled PPEGMA-b-P(DPA-coHEMA)-Ce6 nanoparticles (NPs) with a mean diameter of about 78 nm and polydispersity index of 0.27 were obtained through the dialysis method. The surface potential of PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs can undergo changes from negative (−1.45 mV) at pH 7.4 to highly positive (+11.6 mV) at pH 6.0, as characterized by the zetasizer. The extent of interaction between the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs and bacteria (Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus)), evaluated by flow cytometry and confocal microscopy, was rather limited at pH 7.4. However, the PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs bind bacteria effectively and achieve fluorescence bacterial imaging at pH 6.0. Furthermore, the growth curve of bacteria showed that the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs can achieve good antimicrobial efficacy through combined cationic and photodynamic therapies under acidic pH conditions (pH 6.0). KEYWORDS: pH-sensitive, theranostic nanoparticles, fluorescence imaging, dual-modal antimicrobial therapy

1. INTRODUCTION

develop targeting theranostic antimicrobial agents to combat bacterial infections. Many bacterial infections can be treated effectively if diagnosed accurately at an early stage. Thus, it is critical to detect bacteria rapidly and accurately in clinical practice. Various techniques have been developed for bacterial imaging, such as optoacoustic imaging and optical imaging.7 Fluorescence imaging as one type of optical imaging has many advantages such as being rapid, having a low cost, and being noninvasive. Various types of fluorescence bacterial imaging systems have been developed for accurate bacterial detection.12−14 On the other hand, many strategies have been developed to combat bacterial infections, including antimicrobial cationic polymers,15−18 inorganic nanomaterials,19,20 and photothermal and photodynamic therapy agents.21−23 The use of antimicrobial cationic polymers24−31 is an effective strategy to minimize

Bacterial infections are associated with a variety of diseases and are a major threat to human health. Antibiotic therapy is the main treatment for bacterial infections. However, the emergence of drug-resistant infections has become a widespread problem in public health due to overuse and misuse of the drugs in medicine and agriculture.1,2 In 2017, the World Health Organization (WHO) has for the first time released a list of the drug-resistant bacteria that pose the greatest threat to human health. Therefore, it is urgent to develop new effective antimicrobial strategies to combat bacterial infections. The concept of theranostics was proposed in 2002, and it has been proven to be an effective method to enhance therapeutic efficacy while minimizing side effects of therapeutic agents.3−5 Thus, it is a promising strategy to fabricate theranostic antimicrobial agents to combat bacterial infections effectively by combining bacterial imaging and treatment in a single platform.6,7 On the other hand, bacterial targeting has been widely used to improve bacterial diagnosis accuracy8 and antimicrobial efficacy.9−11 Therefore, it would be desirable to © XXXX American Chemical Society

Received: August 13, 2018 Accepted: October 19, 2018

A

DOI: 10.1021/acsanm.8b01401 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Article

ACS Applied Nano Materials

Scheme 1. Schematic Illustration of the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs as Theranostic Antimicrobial Agents

to single therapy. Although considerable effort has been devoted to cationic polymer therapy and PDT, antimicrobial systems synergetically combining both effects, and with “ondemand” targeting and antimicrobial activity, have yet to be more thoroughly investigated in the treatment of bacterial infections. Herein, a pH-sensitive theranostic nanoparticle system, which was prepared through self-assembly of poly(poly(ethylene glycol) methyl ether methacrylate)-b-poly(2-(diisopropylamino) ethyl methacrylate-co-2-hydroxyethyl methacrylate)-chlorin e6 (PPEGMA-b-P(DPA-co-HEMA)-Ce6), was introduced for on-demand targeting, fluorescence bacterial imaging, and synergetic antimicrobial therapy via combined cationic therapy and PDT (Scheme 1). We expect the nanoparticles to exhibit limited interaction and negligible toxicity to normal tissues during circulation after the nanoparticles have been intravenously injected into the bloodstream. At the bacterial infection sites, bacteria release organic acids, such as lactic and acetic acids, via anaerobic glycolysis,18,45,46 resulting in weakly acidic environment. When the nanoparticles arrive at the acidic bacterial infections sites through blood circulation, the NPs can effectively bind to the bacteria and accumulate at the acidic infection sites. Thus, one can use the fluorescence imaging system to image the infection sites, which in turn can be used to precisely guide the photodynamic therapy, and achieve synergistically cationic and photodynamic therapies. The pH-sensitive P(DPA-co-HEMA)Ce6 chain segment can contribute to bacterial targeting and cationic therapy under acidic conditions (pH 6.0), while photosensitizer Ce6 can emit fluorescence for bacterial imaging and production of ROS for antimicrobial PDT under irradiation. PPEGMA-b-P(DPA-co-HEMA)-Ce6 can self-assemble into core−shell nanoparticles in an aqueous medium, with the hydrophobic P(DPA-co-HEMA)-Ce6 core and the hydrophilic PPEGMA shell. Compared to free Ce6, PPEGMAb-P(DPA-co-HEMA)-Ce6 NPs could target the acidic bacterial infection sites and achieve synergetic therapies. PPEGMA-bP(DPA-co-HEMA)-Ce6 nanoparticles exhibit on-demand pHresponsive bacterial targeting because of the pH-sensitivity of the PDPA chain segment. Under physiological conditions, the PPEGMA-b-P(DPA-co-HEMA)-Ce6 nanoparticles cannot bind to bacteria because of their negatively charged surface. However, they exhibit good bacteria targeting and fluorescence

the spread of infections. The main mechanism of their antimicrobial action is to induce bacterial membrane damage. Cationic polymers can interact with negatively charged bacterial surfaces to compromise the plasma membrane integrity. A large number of cationic antimicrobial polymers have been developed for effective antimicrobial therapy. For instance, Cho et al.26 synthesized a series of guanidine functionalized aliphatic biodegradable polycarbonates which showed strong biocidal activity against a broad spectrum of microorganisms. Cationic peptidopolysaccharides have also been synthesized by “click” chemistry for enhanced broadspectrum antimicrobial activities.27 Photodynamic antimicrobial therapy (PDT)22 is another promising antimicrobial strategy for combating bacterial infections. PDT is based on combining photosensitizers (PS) with light irradiation to trigger cell killing. PS can generate reactive oxygen species (ROS), especially singlet oxygen (1O2), under light irradiation in the presence of oxygen. ROS are mainly responsible for the excellent broad-spectrum antimicrobial activities of PDT, as well as for preventing bacteria from developing drug resistance, by damaging intracellular biomolecules, such as proteins, nucleic acids, and lipids. There are numerous reports in the literature on the antimicrobial application of PDT.32−37 A light-activatable micellar nanocarrier system has been fabricated for eradication of multidrug-resistant staphylococcal infections by photodynamic therapy.33 A surface charge-conversion nanoparticle system has also been developed for photodynamic treatment of urinary tract bacterial infections.34 In addition to these individual therapies, developing antimicrobial agents for dual-modal therapies38−41 is a promising approach to combat bacterial infections as the synergistic effects can enhance therapeutic efficacy. Lipasesensitive liposomes for a skin disorder have been developed by combining photodynamic and antibiotic chemotherapy.42 Cationic polycarbonate-grafted superparamagnetic nanoparticles have been fabricated for the synergistic dual-modal antimicrobial therapy by the cationic shell and magnetic hyperthermia.43 Ruthenium nanoparticles for photodynamic/ photothermal dual-modality phototherapeutic elimination of pathogenic bacteria have also been developed.44 All these studies demonstrated that the dual-modal antimicrobial therapy has enhanced antimicrobial performance compared B


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Briefly, PPEGMA-b-P(DPA-co-HEMA)-Ce6 (10 mg) was dissolved in acetone/dimethyl sulfoxide (0.5 mL, v/v 1:1), and the resulting mixture was then transferred into a dialysis tube (MW cutoff = 3500) and dialyzed against PBS (pH 7.4) for 24 h. The nanoparticle solution was collected and stored at 4 °C. 2.6. Characterization. The molecular weight and dispersity of the polymers were characterized using a Waters’ gel permeation chromatography (GPC) system equipped with a 2414 refractive index detector. THF was used as the mobile phase with a flow rate of 1.0 mL/min at 35 °C. The size and zeta potential of the PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs in PBS at different pH’s were measured on a Malvern Zetasizer (Nano ZS, model ZEN 3600, Malvern Instruments, Worcestershire, UK). UV−vis absorption of the NPs was measured on the Shimadzu spectrophotometer (UV-1601PC). The Shimadzu spectrofluorophotometer (RF-5301PC) was utilized to recorded fluorescence emission spectra of the NPs (excitation wavelength 405 nm). Critical micelle concentration (CMC) of PPEGMA-b-P(DPA-co-HEMA) was determined using nile red as the fluorescence probe according to the procedures reported previously (see Supporting Information for details).48 The morphology of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs was revealed by field emission transmission electron microscopy (JEOL JEM-2100F) after drying a drop of nanoparticle solution on carbon-coated copper grids. 2.7. In Vitro Cytotoxicity of PPEGMA-b-P(DPA-co-HEMA)Ce6 NPs. The cytotoxicity of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs against 3T3 mouse embryonic fibroblast (3T3 cells) was determined by MTT assay. Briefly, 3T3 cells were seeded in 96-well plates at 5 × 103 cells per well, using 100 μL of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and the cells were incubated overnight at 37 °C. Then, the culture medium in each well was replaced by the same volume of DMEM medium in the presence of PPEGMA-b-P(DPA-co-HEMA)Ce6 NPs with different concentrations (0.025−0.5 mg/mL). The cells were incubated for 24 h. The culture medium in each well was then replaced by 100 μL of DMEM medium containing MTT with the concentration of 0.5 mg/mL, and the cells were incubated for an additional 4 h at 37 °C. After 4 h, the MTT-containing medium was carefully replaced with 100 μL of DMSO. After gentle agitation for 10 min, the absorbance at 595 nm of each well was recorded on a microplate reader. All experiments were repeated three times. 2.8. pH-Dependent Interaction of NPs with Bacteria. After being cultured in Tryptic soy broth (TSB) overnight at 37 °C, E. coli cells were collected by centrifugation at 2700 rpm for 5 min at room temperature, followed by washing with sterile phosphate-buffered saline (PBS) twice. The bacterial concentration was determined by measuring optical density (OD) at 600 nm, where OD of 0.5 was equivalent to ∼1 × 109 cells/mL. The bacteria were then suspended in PBS of different pH values with a final concentration of 2 × 108 cells/mL. Solutions of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs (0.5 mL, 0.2 mg/mL) or Ce6 (0.5 mL, 9.34 μg/mL) in different pH buffers were mixed with E. coli suspension (0.5 mL) in sterile 2 mL tubes. After incubation at 37 °C for 3 h, the mixture was centrifuged at 2700 rpm for 5 min at room temperature, washed twice, and suspended in 1 mL of PBS (pH 6.0). Flow cytometry (excitation wavelength: 633 nm, CyAn ADP Analyzer, Beckman Coulter) was utilized to detect the fluorescence signal of Ce6 of bacteria. All experiments were repeated three times. For microscopic studies, 10 μL of this suspension was placed on a microscope glass side, and micrographs were taken on a confocal microscope (Olympus Fluoview FV1000 TIRF) at an excitation wavelength of 633 nm. The pH-dependent interaction of the PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs with S. aureus was also investigated by confocal microscope. All experiments were repeated three times. 2.9. Antimicrobial Activity of NPs. The bacteria were suspended in PBS of different pH values with a final concentration of 1 × 105 cells/mL. The PPEGMA-b-P(DPA-co-HEMA)-Ce6 solution (0.03 mg/mL) in buffers of different pH values were mixed with the E. coli suspension at a volume ratio of 2:1. A 1 mL portion of the mixture was transferred to a 5 mL glass vial. The vial in

imaging in an acidic environment (pH 6.0) as their surface become positively charged due to protonation of DPA and negative charge of the bacterial surface. In addition, PPEGMAb-P(DPA-co-HEMA)-Ce6 can achieve “on-demand” combined cationic and photodynamic antimicrobial therapy under light irradiation in an acidic environment (pH 6.0).

2. EXPERIMENTAL SECTION 2.1. Materials. Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn = 500), 2-(diisopropylamino)ethyl methacrylate (DPA, 97%), 2-hydroxyethyl methacrylate (HEMA, 97%), dioxane (99.8%), oxalyl chloride (98%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 98%), tryptic soy broth (TSB), and mueller hinton broth (MHB) were purchased from Sigma-Aldrich Chemical Co. Chlorin e6 (Ce6) was purchased from Frontier Scientific, Inc. Escherichia coli (E. coli, ATCC25922), Staphylococcus aureus (S. aureus, ATCC25923), and 3T3 mouse embryonic fibroblast (3T3 cells) were purchased from American Type Culture Collection (Manassas, VA). Nile red was purchased from Tokyo Chemical Industry Co., Ltd. PEGMA was passed through basic alumina columns to remove the inhibitors prior to use. 4-Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDTPA) was prepared according to procedures reported previously (see Supporting Information and Figure S1 for details).47 2.2. Preparation of Poly(poly(ethylene glycol) methyl ether methacrylate) (PPEGMA). PPEGMA was prepared by reversible addition−fragmentation chain transfer (RAFT) polymerization. PEGMA (2.41 g, 4.82 mmol), CDTPA (97.2 mg, 0.24 mmol), and 2,2′-azobis(2-methylpropionitrile) (AIBN, 7.9 mg, 0.048 mmol) were dissolved in dioxane (5 mL), and the resulting solution was degassed by purging with argon for 30 min. The polymerization was allowed to proceed at 70 °C for 24 h. After polymerization, the reaction mixture was poured into cold diethyl ether, and PPEGMA was obtained as the macro-RAFT chain transfer agent after drying under reduced pressure. Yield: 81%. 2.3. Preparation of Poly(poly(ethylene glycol) methyl ether methacrylate)-b-poly(2-(diisopropylamino) ethyl methacrylate-co-2-hydroxyethyl methacrylate) (PPEGMA-b-P(DPA-coHEMA)). Typically, PPEGMA (1 g), DPA (1.91 g, 9 mmol), HEMA (0.13 g, 1 mmol), and AIBN (6.5 mg, 0.04 mmol) were dissolved in dioxane (6 mL), and the resulting solution was degassed by purging with argon for 30 min. The polymerization reaction was allowed to proceed at 70 °C for 24 h. After polymerization, the mixture was dialyzed against acetone for 2 days and deionized (DI) water for 1 day using a dialysis membrane (MW cutoff = 3500). The final product, PPEGMA-b-P(DPA-co-HEMA), was obtained upon lyophilization. Yield: 72%. 2.4. Preparation of Poly(poly(ethylene glycol) methyl ether methacrylate)-b-poly(2-(diisopropylamino) ethyl methacrylate-co-2-hydroxyethyl methacrylate)-chlorin e6 (PPEGMA-bP(DPA-co-HEMA)-Ce6). Chlorin e6 (Ce6, 20 mg, 0.03 mmol) was dissolved in dichloromethane (DCM, 10 mL), and oxalyl chloride (84 μL) and two drops of dimethylformamide (DMF) were added at 0 °C. The mixture was stirred for 3 h at room temperature, and the solvent was removed by rotary evaporation. DCM (10 mL) was added, and the resulting mixture was dropped into the PPEGMA-bP(DPA-co-HEMA) solution (0.1 g in 40 mL DCM) containing trimethylamine (TEA, 13.9 μL). The reaction was allowed to proceed overnight at room temperature, and the solvent was removed under reduced pressure. Tetrahydrofuran (THF, 20 mL) was added, and the resulting mixture was dialyzed against ethanol/deionized water for 3 days and deionized water for 1 day using a dialysis membrane (MW cutoff = 3500). The final product, PPEGMA-b-P(DPA-co-HEMA)Ce6, was obtained by lyophilization. Yield: 85%. The content of Ce6 in PPEGMA-b-P(DPA-co-HEMA)-Ce6 was determined by UV−vis absorption at 405 nm and was calculated as follows: Content of Ce6 (wt %) = (weight of Ce6/weight of polymer) × 100%. 2.5. Preparation of Nanoparticles. PPEGMA-b-P(DPA-coHEMA)-Ce6 nanoparticles were prepared by the dialysis method. C


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Figure 1. Synthetic route for the preparation of PPEGMA-b-P(DPA-co-HEMA)-Ce6. the upright position on a flat surface was irradiated horizontally with a laser (660 nm, 100 mW cm−2, laser spot size of 4 cm2, Beijing Laserwave Optoelectronics Technology Co., Ltd.) for 10 min. After culturing at 37 °C for 2 h, the solution (100 μL) was introduced into TSB (2 mL). The mixture was vortexed for 10 s, and 100 μL of the mixture was added to a 96-well plate. The plate was incubated at 37 °C, and the time-dependent OD at 630 nm was recorded on a microplate reader (BioTek Instruments ELX800). All experiments were repeated three times. The antimicrobial activity of PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs against S. aureus was investigated using the same method. The PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs solution with a concentration of 0.5 mg/mL was used. All experiments were repeated three times.

PPEGMA-b-P(DPA-co-HEMA). Gel permeation chromatography (GPC) traces of PPEGMA and PPEGMA-b-P(DPA-coHEMA) (Figure S3) showed a clear shift to a lower retention time of the latter, consistent with successful diblock copolymerization. The average molecular weight and dispersity of polymers are summarized in Table 1. The molecular weight Table 1. Molecular Weight and Polydispersity of the Polymers sample

Mn (1H NMR)a

Mn (GPC)b

Mw/Mnb

PPEGMA PPEGMA-b-P(DPA-co-HEMA)

9500 20 213

7585 14 924

1.28 1.65

a

Determined from 1H NMR. bDetermined from GPC (eluent, THF; flow rate, 1 mL/min; standards, polystyrene).

3. RESULTS AND DISCUSSION 3.1. Preparation of Poly(poly(ethylene glycol) methyl ether methacrylate)-b-poly(2-(diisopropylamino) ethyl methacrylate-co-2-hydroxyethyl methacrylate)-chlorin e6 (PPEGMA-b-P(DPA-co-HEMA)-Ce6). To fabricate pHsensitive theranostic nanoparticles for on-demand targeting, fluorescence bacterial imaging, and synergetic antimicrobial therapies, PPEGMA-b-P(DPA-co-HEMA)-Ce6 was prepared through reversible addition−fragmentation chain transfer (RAFT) polymerization and post-modification (Figure 1). At first, poly(poly(ethylene glycol) methyl ether methacrylate), PPEGMA, was obtained by RAFT polymerization of poly(ethylene glycol) methyl ether methacrylate. Then, the diblock copolymer, PPEGMA-b-P(DPA-co-HEMA), was prepared by RAFT polymerization of 2-(diisopropylamino)ethyl methacrylate and 2-hydroxyethyl methacrylate utilizing PPEGMA as the macromolecular chain transfer agent. The chemical structure of PPEGMA and PPEGMA-b-P(DPA-co-HEMA) was characterized by 1H NMR spectroscopy. Figure S2 clearly shows the characteristic chemical shifts assigned to PPEGMA (hydrogen protons of (−OCH2CH2−) at 3.65 ppm) and PDPA (hydrogen protons of (CH 3 )2 −CHN− at 2.62 ppm), demonstrating the successful synthesis of PPEGMA and

dispersity of PPEGMA-b-P(DPA-co-HEMA) increased slightly to around 1.65, compared to 1.28 for PPEGMA. This increase is probably caused by a small number of terminated chains, as tailing can be detected from the GPC elution curves. Finally, Ce6 chloride, which was obtained through reaction between Ce6 and oxalyl chloride, was conjugated to the hydroxyl group of PPEGMA-b-P(DPA-co-HEMA) to form the final product PPEGMA-b-P(DPA-co-HEMA)-Ce6. The successful synthesis of PPEGMA-b-P(DPA-co-HEMA)-Ce6 was ascertained by 1H NMR spectroscopy, and the chemical shifts of Ce6 located at 6−6.5 and 8.5−10 ppm can be clearly observed (Figure 2). In addition, PPEGMA-b-P(DPA-co-HEMA)-Ce6 was dissolved in DMF which is a good solvent for the nanoparticle. No hydrodynamic diameters of the nanoparticles were discernible in dynamic light scattering (DLS) measurement of the solution, indicating that the cross-link reaction probably did not occur between Ce6 and PPEGMA-b-P(DPA-co-HEMA). The content of Ce6 in PPEGMA-b-P(DPA-co-HEMA)-Ce6 (weight of Ce6/weight of polymer) was measured to be 4.65 wt % from UV−vis absorption at 405 nm. This Ce6 content D


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Figure 2. 1H NMR spectrum of PPEGMA-b-P(DPA-co-HEMA)-Ce6 in CDCl3/DMSO-d6 (10:3, v/v). The chemical shifts of Ce6 are located at 6−6.5 and 8.5−10 ppm.

application. The cytotoxicity of PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs against 3T3 mouse embryonic fibroblast (3T3 cells) was investigated in the absence of light irradiation through MTT assay, which was employed to prove biocompatibility of the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs in blood circulation and in normal tissues. The results are shown in Figure 4. There was almost no toxicity detected among the tested concentrations (from 25 to 500 μg/mL), indicating good biocompatibility of the PPEGMA-b-P(DPAco-HEMA)-Ce6 NPs. Reactive oxygen species (ROS) generation upon light-activation of the PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs under different pH conditions was investigated using 9,10-anthracenediyl-bis(methylene)-dimalonic acid (ADA) as the 1O2 probe. ADA can trap ROS, leading to a decrease in ADA absorption at 400 nm. The results are shown in Figure S5, and there was no decrease in absorption in the absence of light irradiation. This observation suggested that the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs did not produce ROS without irradiation. In the presence of light irradiation, the absorption of ADA decreased significantly both at pH 7.4 and 6.0, and there was no obvious difference at these two pH’s. Thus, the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs can generate ROS effectively and exhibit similar ROS production efficiency at pH 7.4 and 6.0. Due to pH-sensitivity of the PDPA chain segment, the surface charge of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs was expected to experience a change from negative to positive with decreasing pH. As such, the zeta potentials of PPEGMAb-P(DPA-co-HEMA)-Ce6 NPs at pH’s from 7.4 to 6.0 were investigated. Figure 5 shows that the PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs have a zeta potential of −1.45 mV at pH 7.4. However, the zeta potential of PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs gradually became positive with decreasing pH. The zeta potential of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs changed to almost neutral (+0.08 mV) at pH 7.0, and further increased to +11.6 mV at pH 6.0. The surface charge-

suggested that approximate two molecules of Ce6 conjugated with each PPEGMA-b-P(DPA-co-HEMA) chain. 3.2. Preparation and Characterization of PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs. To make sure whether PPEGMA-b-P(DPA-co-HEMA)-Ce6 can self-assemble into nanoparticles in an aqueous medium, critical micelle concentration (CMC) of PPEGMA-b-P(DPA-co-HEMA) was investigated utilizing nile red as the fluorescence probe, and it was determined to be 29.4 μg/mL (Figure S4). PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs were obtained through the dialysis method and had a mean diameter of about 78 nm and polydispersity index of 0.27 in PBS of pH 7.4, as determined by DLS (Figure 3a). Transmission electron microscopy (TEM) was utilized to characterize the morphology of NPs, as shown in Figure 3b. The TEM result reveals that the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs were almost spherical in shape and had a mean diameter of about 65 nm. It was slightly smaller than that determined by DLS measurement, probably due to shrinkage of the nanoparticles upon drying during sample preparation for TEM measurements. The optical properties of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs were also investigated as they are important criteria for a fluorescent theranostic agent. Figure 3c shows that PPEGMAb-P(DPA-co-HEMA)-Ce6 NPs give rise to intense absorptions at 405 and 675 nm. Compared to Ce6, the absorption peak at 675 nm of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs is redshifted, and this can be attributed to the typical response of Ce6 in different chemical environments.49 In addition, similar to Ce6, the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs exhibit an intense fluorescence emission peak at 660 nm, which would facilitate the fluorescence bacterial imaging (Figure 3d). This result indicated that conjugation of Ce6 to PPEGMA-bP(DPA-co-HEMA) did not affect the fluorescence of Ce6 appreciably. The biocompatibility of an antimicrobial agent is an important parameter that determines its suitability for in vivo E


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Figure 3. (a) Size distribution of the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs determined by DLS at pH 7.4. (b) TEM image of the PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs. Scale bar is 200 nm. (c) UV−vis absorption of Ce6 and the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs. (d) Fluorescence emission spectra of Ce6 and the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs. Excitation wavelength: 405 nm.

Figure 4. Cytotoxicity of the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs against 3T3 cells in MTT assay. The incubation time was 24 h. Mean ± standard deviation shown (n = 3).

Figure 5. Zeta potential of the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs (0.5 mg/mL) at different pH’s determined by DLS. Mean ± standard deviation shown (n = 3).

reversal can be attributed to the protonation of PDPA chain segment. The size of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs under different pH conditions was also investigated. The size of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs decreases slightly

from pH 7.4 to 7.0, and then increases to about 90 nm with pH decreasing to 6.0 (Figure S6). 3.3. pH-Dependent Interaction of PPEGMA-b-P(DPAco-HEMA)-Ce6 NPs with Bacteria. Due to pH-sensitivity of F


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NPs are slightly negatively charged and the PPEGMA segments on the surface of nanoparticles inhibit the interaction between the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs and E. coli with negative surface charge at pH 7.4. At pH 6.0, the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs become positively charged because of protonation of the PDPA chains, resulting in a strong interaction between the nanoparticles and E. coli. To provide visual confirmation of the binding of PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs to E. coli, confocal microscopy was also used to investigate their interaction under different pH conditions (Figure 7). Compared to the physiological condition (pH 7.4), E. coli at pH 6.0 gives rise to intense fluorescence signal of Ce6, confirming the pH-sensitive binding of the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs to E. coli. These results indicate that the PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs exhibit effective on-demand binding to E. coli and fluorescence imaging under acidic conditions. Similar pH-dependent binding of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs to S. aureus was observed (Figure S7). 3.4. Dual-Modal Antimicrobial Activity of the Nanoparticles. The PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs are expected to provide synergetic antimicrobial therapy through cationic P(DPA-co-HEMA)-Ce6 and photodynamic therapy. At first, the minimum inhibitory concentration (MIC) of the PPEGMA-b-P(DPA-co-HEMA) and PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs against E. coli and S. aureus was investigated in the absence of light (660 nm) irradiation. MIC was defined as the minimum NPs concentration at which no bacterial growth was detected in a turbidity assay. As shown in Table S1, the PPEGMA-b-P(DPA-co-HEMA) NPs and PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs could not inhibit the growth of E. coli up to the highest concentration used (1024 μg/mL) at pH 7.4. However, the PPEGMA-b-P(DPA-co-HEMA) NPs exhibit a MIC of 256 μg/mL while the MIC of PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs was 32 μg/mL at pH 6.0. For S. aureus, the PPEGMA-b-P(DPA-co-HEMA) NPs could not inhibit their growth up to the highest concentration used

the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs, differential binding of the NPs to bacteria can be expected under the acidic microenvironments of bacteria. As Gram-negative bacteria can cause severe infections,50 E. coli was selected as model bacteria to evaluate the interaction between PPEGMAb-P(DPA-co-HEMA)-Ce6 NPs and bacteria under different pH conditions in flow cytometry. As shown in Figure 6, high

Figure 6. Interaction of the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs and Ce6 with E. coli in PBS of pH ranging from 6.0 to pH 7.4 for 3 h. The interaction of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs and Ce6 with E. coli was measured by flow cytometry, and the excitation wavelength was 633 nm. Mean ± standard deviation shown (n = 3).

fluorescence signals were observed from E. coli after treatment with the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs under low pH conditions. However, low fluorescence signal intensities were observed from E. coli after incubation with Ce6 alone. This result indicates a strong interaction between PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs and E. coli at low pH. The possible reason is that PPEGMA-b-P(DPA-co-HEMA)-Ce6

Figure 7. Fluorescent confocal microscopy of E. coli after incubation with the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs (0.1 mg/mL) under different pH conditions for 3 h. For each panel, the images from left to right show the bright field of bacteria, Ce6 fluorescence (red), and the overlay of two images. Scale bar is 20 μm. G


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ACS Applied Nano Materials (8000 μg/mL) at both pH 7.4 and 6.0. However, the PPEGMA-b-P(DPA-co-HEMA)-Ce6 has a MIC of 1024 μg/ mL at pH 6.0, whereas the growth of S. aureus cannot be inhibited up to the highest concentration used (1024 μg/mL) at pH 7.4. As PPEGMA cannot inhibit the growth of bacteria, the antimicrobial effect of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs comes from the P(DPA-co-HEMA)-Ce6 chain segment. These results indicate that the PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs have antimicrobial effects on E. coli and S. aureus when the P(DPA-co-HEMA)-Ce6 chains are protonated at pH 6.0. Furthermore, conjugation of Ce6 enhances the antimicrobial ability of the P(DPA-co-HEMA) chain segment. The synergetic antimicrobial activity of the PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs was evaluated from the growth curve of E. coli at pH 7.4 and 6.0 in the presence of PPEGMAb-P(DPA-co-HEMA)-Ce6 NPs (0.02 mg/mL) with and without light (660 nm) irradiation. The growth of bacteria was determined by measuring OD values at 630 nm. As shown in Figure 8a, the growth of E. coli was not affected at pH 7.4

the bacteria in the absence of light irradiation at pH 6.0 at the NPs concentration of 0.02 mg/mL, and slow down the growth of bacteria in the first 10 h. Moreover, the growth of E. coli was completely inhibited at pH 6.0 under light irradiation. As the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs exhibit similar ROS generation ability under light irradiation at pH 7.4 and 6.0 (Figure S5), the difference in PDT efficacy of PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs against E. coli probably comes from the different interaction behavior between the NPs and E. coli at different pH values. The P(DPA-co-HEMA)-Ce6 segments are in the deprotonated state at pH 7.4, which leads to limited interaction of the PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs with E. coli at pH 7.4. As a result, ROS produced by PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs under light irradiation cannot inhibit the growth of E. coli under the low interaction conditions because of the short lifetime and diffusion distance of ROS.51 However, P(DPA-co-HEMA)-Ce6 chains become protonated under the acidic condition (pH 6.0), which can enhance the interaction between E. coli and the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs. The enhanced interaction between E. coli and the PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs allows the light generated ROS to eliminate E. coli effectively. Hence, the growth of E. coli can be completely inhibited in the presence of PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs under light irradiation in an acidic environment (pH 6.0). Similar pH-dependent synergetic antimicrobial therapy of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs against S. aureus was observed (Figure S8). However, PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs have a lower antimicrobial efficacy for S. aureus than for E. coli, and a high dose of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs is necessary.

4. CONCLUSION A pH-responsive theranostic system was fabricated for bacteria targeting, fluorescence imaging, and synergetic cationic and photodynamic antimicrobial therapy. PPEGMA-b-P(DPA-coHEMA)-Ce6 NPs of about 78 nm in size were obtained by self-assembly of the amphiphilic polymer, PPEGMA-b-P(DPAco-HEMA)-Ce6, in aqueous solution. The surface charge of PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs was slightly negative under physiological conditions, but can become highly positive in an acidic environment (pH < 7.0). The PPEGMA-bP(DPA-co-HEMA)-Ce6 NPs do not interact with bacteria (E. coli and S. aureus) at pH 7.4. However, upon surface chargereversal at pH < 7.0, the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs can bind to bacteria (E. coli and S. aureus) effectively and achieve fluorescence imaging of the bacteria in the acidic environment of bacterial culture. Furthermore, the PPEGMAb-P(DPA-co-HEMA)-Ce6 NPs can inhibit the growth of bacteria (E. coli and S. aureus) in an acidic environment under light irradiation through combined cationic and photodynamic therapies. These results indicate that the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NP is a potential tharanostic system for targeting, fluorescence imaging, and dualmodal antimicrobial therapy.

Figure 8. Growth curve of E. coli (1 × 105 cells/mL) after incubation with PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs (0.03 mg/mL) at a volume ratio of 2:1 at (a) pH 7.4 and (b) pH 6.0 with and without light irradiation (660 nm, 100 mW/cm2) for 10 min.

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ASSOCIATED CONTENT

S Supporting Information *

whether under light irradiation or not. However, the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs were found to slow down the growth of E. coli at pH 6.0 in the absence of light irradiation in the first 10 h, and the bacteria growth eventually recovered to the same level as the control (Figure 8b). Thus, the PPEGMA-b-P(DPA-co-HEMA)-Ce6 NPs can kill some of

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b01401. Synthesis and experimental details and additional characterization data (PDF) H


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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (E.-T.K). ORCID

Li Qun Xu: 0000-0002-6780-114X Koon-Gee Neoh: 0000-0002-2700-1914 En-Tang Kang: 0000-0003-0599-7834 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors would like to acknowledge the financial support of this study from the Singapore Millennium Foundation under Grant 1123004048 (NUS WBS R279-000-428-592).

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