Cholesterol-Assisted Bacterial Cell Surface Engineering for

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Cholesterol-Assisted Bacterial Cell Surface Engineering for Photodynamic Inactivation of Gram-Positive and Gram-Negative Bacteria Hao-Ran Jia, Ya-Xuan Zhu, Zhan Chen, and Fu-Gen Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 20 Apr 2017 Downloaded from http://pubs.acs.org on April 23, 2017

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ACS Applied Materials & Interfaces

Cholesterol-Assisted Bacterial Cell Surface Engineering for Photodynamic Inactivation of Gram-Positive and Gram-Negative Bacteria

Hao-Ran Jia,†,§ Ya-Xuan Zhu,†,§ Zhan Chen,*,‡ and Fu-Gen Wu*,†

†

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering,

Southeast University, Nanjing 210096, P. R. China ‡

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor,

Michigan 48109, United States

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ABSTRACT Antibacterial photodynamic therapy (PDT) which enables effective killing of regular and multidrug-resistant (MDR) bacteria is a promising treatment modality for bacterial infection. However, because most photosensitizer (PS) molecules fail to strongly interact with the surface of Gram-negative bacteria, this technique is only suitable for treating Gram-positive bacterial infection, which largely hampers its practical applications. Herein, we revealed for the first time that cholesterol could significantly facilitate the hydrophobic binding of PSs to the bacterial surface, achieving the hydrophobic interaction-based bacterial cell surface engineering that could effectively photo-inactivate both Gram-negative and Gram-positive bacteria. An amphiphilic polymer composed of a polyethylene glycol (PEG) segment terminated with protoporphyrin IX (PpIX, an anionic PS) and cholesterol was constructed (abbreviated as Chol-PEG-PpIX), which could self-assemble into micelle-like nanoparticles (NPs) in aqueous solution. When encountering the Gram-negative Escherichia coli cells, the Chol-PEG-PpIX NPs would dis-assemble and the PpIX moieties could effectively bind to the bacterial surface with the help of the cholesterol moieties, resulting in the significantly enhanced fluorescence emission of the bacterial surface. Under white light irradiation, the light-triggered singlet oxygen (1O2) generation of the membrane-bound PpIX could not only severely damage the outer membrane, but also facilitate the entry of external Chol-PEG-PpIX into the bacteria, achieving more than 99% bactericidal efficiency. Besides, as expected, the Chol-PEG-PpIX NPs also exhibited excellent antibacterial performance against the Gram-positive Staphylococcus aureus. We also verified that this nanoagent possesses negligible dark cytotoxicity towards mammalian cells and good hemocompatibility. To the best of our knowledge, this study demonstrates for the first time the feasibility of constructing a fully hydrophobic interaction-based and outer membrane-anchored antibacterial PDT nanoagent. KEYWORDS: polymeric nanoparticle, outer membrane binding, hydrophobic interaction, photodynamic inactivation, bacterial cell surface modification

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1. INTRODUCTION Over the past few years, the growing emergence of multidrug-resistant (MDR) bacterial strains has drastically threatened human health.1 The effectiveness of conventional antibiotics against MDR bacteria is decreasing;2 the situation is even exacerbated due to the indiscriminate use of antibiotics in medical treatment.3 Therefore, it is of great urgency to develop new and potent antibacterial strategies. Antimicrobial photodynamic therapy (PDT) typically involves a photosensitizer (PS), light, and molecular oxygen to generate reactive oxygen species, primarily singlet oxygen (1O2), and has been reported as an alternative strategy to inhibit bacteria growth.4 The highly oxidative 1O2 can cause irreversible damage to many important biomolecules such as membrane lipids,5 proteins,6 and cellular DNAs,7 which ultimately leads to bacterial cell death. In addition, MDR bacteria have shown to be as vulnerable as non-MDR bacteria to PDT,8,9 which offers tremendous promise for PDT to combat drug resistance. However, PDT has very different effects on Gram-positive and Gram-negative bacteria because of their different membrane structures.10 In most cases, PSs can penetrate through the relatively porous cell walls of Gram-positive bacteria and effectively kill bacteria upon light irradiation. For Gram-negative bacteria, due to the presence of outer membranes, they display robust resistance to various photosensitizers.8,10 This highly organized membrane structure of Gram-negative bacteria acts as a formidable defensive barrier to numerous drug molecules.4 To solve this problem, one approach is to use outer membrane-disrupting agents, such as the

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lipopeptide antibiotic polymixin B11 and ethylenediaminetetraacetic acid (EDTA),12 to increase the susceptibility of bacteria to PDT. However, the adoption of such pretreatment may raise concerns regarding the potential toxicity and tissue reaction of these agents. For example, EDTA has been reported to show a cytotoxic effect towards mammalian cells in a dose-dependent manner13 and may cause tissue inflammation at relatively high local concentrations.14 Recently, cell surface engineering as a versatile technique has received increasing attention to modify cell plasma membranes and endow the cells with new functions and properties.15 Commonly used approaches for cell surface engineering include covalent conjugation,16 hydrophobic interaction,17 and electrostatic interaction.15 Cell surface engineering has been widely used for many biomedical applications, including plasma membrane imaging,18−24 extracellular microenvironment detection,25 diabetes treatment,26 cancer theranostics,27−32 and infectious disease treatment.33−34 To employ cell surface engineering for PDT-based antibacterial application, the electrostatic interaction between the negatively charged bacterial surface and cationic PSs35−40 and positively charged PDT nanoparticles (NPs), such as conjugated polymer-based NPs,41−44 supramolecular nanoassemblies,45−47 carbon dots,48 and fullerenes,49 is widely adopted to achieve satisfying therapeutic outcome. Unfortunately, the electrostatic interaction between cationic reagents/NPs and bacterial surface may be affected by changes in the ionic strength or environmental pH of the aqueous solution,18,50 which may possibly lead to the detach of the reagents/NPs from the bacterial surface and the compromised PDT efficacy. Moreover, most positively

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charged materials were found to be highly toxic to mammalian cells,51 especially red blood cells,52 and may cause immunogenic reactions.53 Alternatively, diverse amphiphilic polymers were designed to achieve effective surface modification of mammalian cells through hydrophobic interactions between their anchoring groups (e.g., alkyl chains and lipids) and the plasma membranes, without affecting cell viability.54−56 However, owning to the inherently different structures between bacterial cell surface and mammalian cell surface, many hydrophobic membrane probes that can effectively bind to mammalian cells usually fail to interact with the bacterial surfaces.57,58 Although in some studies, alkyl chains were introduced on cationic PSs or NPs to reinforce the interaction with the bacterial surface,59,60 the cationic moieties were still indispensable for the successful binding. Therefore it is worth exploring the possibility of constructing a safe and potent antibacterial PDT agent that interacts with the bacterial surface solely based on hydrophobic interaction. However, to the best of our knowledge, few reports on such investigation have been published. In this work we designed a noncationic PDT nanoagent which achieves effective PS loading on bacterial surface and excellent antibacterial efficacy (Scheme 1). Through one-step chemical conjugation, an anionic photosensitizer protoporphyrin IX (PpIX) was linked to a cholesterol-modified polyethylene glycol polymer (Chol-PEG) to give an amphiphilic compound Chol-PEG-PpIX. The obtained Chol-PEG-PpIX is highly soluble and can form core-shell nanostructure in aqueous solutions. The nanoagent enables effective binding to the outer membranes of Gram-negative Escherichia

coli

(E.

coli),

despite

its

noncationic

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character

and


antifouling/antiadsorption properties of the PEG chain. We discovered that the cholesterol moiety in Chol-PEG-PpIX greatly facilitated the hydrophobic anchoring of PpIX onto the bacterial outer membrane, resulting in a PpIX-enriched bacterial surface. Therefore, Chol-PEG-PpIX NPs can severely damage the integrity of bacterial outer membranes under mild light irradiation and realize light-regulated increase in the permeability of the outer membranes, which leads to the ultimate cell death. Effective photodynamic inactivation against Gram-positive Staphylococcus aureus (S. aureus) was also achieved. Taken together, we demonstrated the feasibility of achieving fully hydrophobic anchoring-based PS enrichment on the surface of Gram-negative bacteria for improved PDT efficacy, which may shed new light on bacterial cell surface engineering and the design of next-generation antimicrobial agents.

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Scheme 1. Schematic Illustration of Photodynamic Inactivation Against E. coli by Chol-PEG-PpIX upon Light Irradiation.

2. EXPERIMENTAL DETAILS 2.1. Materials. Methoxyl PEG2000 amine (OMe-PEG2000-NH2) was purchased from Jenkem Technology Co., Ltd. Cholesterol-PEG2000-NH2 was obtained from Nanocs, Inc. PpIX, N-hydroxysuccinimide (NHS), and dimethyl sulfoxide (DMSO) were purchased from Aladdin Chemistry Co., Ltd. 1-Ethyl-3-(3-(dimethyl-amino)propyl)carbodimide (EDC) was bought from Sigma-Aldrich. Dialysis membranes with the molecular weight cut-off (MWCO) of 2 kDa were obtained from SpectrumLabs. E. coli total lipid extract was purchased from Avanti Polar Lipids. Singlet oxygen sensor green (SOSG) was ordered from Invitrogen. Cell counting kit-8 (CCK-8) was obtained from Beyotime Institute Biotechnology. 2.2. Synthesis of Chol-PEG-PpIX. To synthesize Chol-PEG-PpIX, 3.37 mg PpIX, 2.78 7



mg EDC, and 4.14 mg NHS were dissolved in DMSO, respectively, and mixed together to react for 1 h at room temperature. Then, the activated PpIX was added to 2.30 mg cholesterol-PEG2000-NH2 (predispersed in DMSO) for further reaction under stirring overnight. The mixture was dialyzed (MWCO = 2 kDa) against DMSO for 3 days and against H2O for 1 day successively. Finally, the obtained Chol-PEG-PpIX was lyophilized and stored at –20 oC for further use. As control, the PEGylated PpIX molecule without cholesterol moiety (OMe-PEG-PpIX) was synthesized via the reaction between PpIX and OMe-PEG2000-NH2 following the similar protocol described above. 2.3. Characterization of Chol-PEG-PpIX Assemblies. The size and morphology of Chol-PEG-PpIX in aqueous solutions were characterized by TEM. Typically, the sample solution (200 µg/mL) was deposited onto a glow-discharged carbon-coated grid, using 2% phosphotungstic acid for negative staining. After being dried at room temperature, the sample was observed using a transmission electron microscope (JEM-2100, JEOL Ltd., Japan). The hydrodynamic diameters of different samples were determined through DLS using a Zetasizer instrument (Nano ZS, Malvern Instruments, United Kingdom). The UV−vis absorption spectra of free PpIX and Chol-PEG-PpIX were measured by a Shimadzu UV-2600 spectrophotometer. 2.4. Preparation of Model Bacterial Membranes. To prepare bacterial membrane-mimic vesicles (liposomes), 4 mg of E. coli total lipid extract powder was hydrated with 2 mL of a 0.9% NaCl solution to the concentration of 2 mg/mL. The lipid suspension was vortexed for 2 min and sonicated for a total of 2 min-pulse period using a tip sonicator at 30 W with a 6 s-interval (4 s-pulse on, 2 s-pulse off).

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2.5. Measurements of Fluorescence Emission and 1O2 Generation. To evaluate the enhanced fluorescence of Chol-PEG-PpIX when anchoring onto the membrane, 1 mL of liposome solution (2 mg/mL) was mixed with 1 mL of Chol-PEG-PpIX solution (10 µM of PpIX) and the formed liposome–Chol-PEG-PpIX mixture was incubated at room temperature for 15 min. Fluorescence spectra of the liposome–Chol-PEG-PpIX mixtures were recorded using a spectrofluorophotometer (RF-5301PC, Shimadzu, Japan). The generation of 1O2 was measured using the SOSG as the indicator. To begin with, 1 µL of SOSG solution was mixed with 2 mL of Chol-PpIX-PEG and liposome–Chol-PEG-PpIX solution, respectively. Then, the mixture was irradiated by a laser (635 nm) at the power density of 14 mW/cm2 and the fluorescence spectra of SOSG were recorded at different time points. 2.6. Bacterial Cell Culture. E. coli and S. aureus bacteria were cultured in lysogeny broth (LB) medium under shaking (180 rpm) at 37 oC. The concentration of bacteria was determined by measuring the optical density at 600 nm (OD600) via UV–vis spectroscopy. 2.7. Confocal Imaging. The bacterial samples for confocal imaging experiments were prepared as follows: When the OD600 value of bacterial solution reached 0.5, 1 mL of bacterial solution was transferred to an Eppendorf tube and centrifuged at 5000 rpm for 5 min. After washing with 0.9% NaCl solutions, the bacteria were re-suspended in 100 µL of Chol-PEG-PpIX solution (10 µM in 0.9% NaCl solution) and incubated under shaking for 15 min. Then, 10 µL of bacterial solution was dropped onto a glass slide and covered by a coverslip. Confocal fluorescence images of stained bacterial cells were obtained on an inverted confocal laser scanning microscope TCS SP8 (Leica, Germany) with a 100× oil

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immersion objective. The 552 nm laser was set to excite the sample and the fluorescence emission was detected in the wavelength range of 600−670 nm. 2.8. Flow Cytometry. E. coli cells were harvested and treated with 10 µM Chol-PEG-PpIX in deionized water, a 10 mM NaCl solution, a 0.9% NaCl solution, and a cell PBS solution, respectively. After incubation at 37 oC for 15 min, the treated E. coli cells were analyzed using a flow cytometer (NovoCyte 2060, ACEA, USA). Channel used for this assay was PerCP with the excitation at 488 nm. 2.9. Antibacterial Activity Measurement. Bacteria were introduced to the fresh LB medium and cultured at 37 oC under shaking until the OD600 value of bacterial solution reached 0.5. Afterwards, 1 mL of the bacterial solution was centrifuged at 5000 rpm for 5 min and the bacterial pellet was washed with 0.9% NaCl solution twice, followed by resuspension in 1 mL of 0.9% NaCl solution. Then, 90 µL of Chol-PEG-PpIX solution at various concentrations was mixed with 10 µL of bacterial suspensions to reach the final PpIX concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 µM, respectively. After incubation for another 30 min, the treated bacteria were irradiated by a white light. The emission spectrum of the white light source was measured using a SpectraScan® Spectroradiometer (PR-655, Photo Research, USA). Specifically, the power, power density, spot size, energy, energy density, and irradiation time were set as 200 mW, 5 mW/cm2, 40 cm2, 360 J, 9 J/cm2, and 30 min, respectively. Next, 50 µL of diluted bacterial solutions was plated on LB agar plates and the number of bacterial colonies was counted and recorded after incubation for 24 h. 2.10. Cytotoxicity Evaluation. L02 cells were cultured in Dulbecco’s modified Eagle’s

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medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 100 IU/mL penicillin–streptomycin. To evaluate the cytotoxicity of Chol-PEG-PpIX, L02 cells were seeded into 96-well plates at a density of 5 × 103 cells per well and incubated for 24 h. Then, the cells were treated with Chol-PEG-PpIX at different concentrations (1, 2, 5, 10, 15, 20, and 30 µM). After incubation for another 24 h, the cell viability was determined by the CCK-8 assay. The absorbance of CCK-8 was recorded using a microplate reader (Thermo-Scientific, Multiskan FC, USA) at the wavelength of 450 nm. 2.11. Hemolysis Rate Test. Hemolysis assay was conducted according to the previously reported procedure.60 Whole blood was collected from mice and preserved in tubes containing sodium citrate. To isolate RBCs, blood was centrifuged three times and re-suspended in a PBS solution. Then, 0.5 mL of RBC solution was mixed with 0.5 mL of Chol-PEG-PpIX solution to give a final Chol-PEG-PpIX concentration of 1, 5, 10, 20, and 50 µM, respectively. RBCs incubated with PBS solutions and ultrapure water were set as negative control and positive control, respectively. The mixture was incubated at 37 oC for 1 h, followed by centrifugation at 4000 rpm for 5 min. The percentage of hemolysis was calculated using the following equation: Hemolysis% = (sample absorbance – negative control absorbance) / (positive control absorbance – negative control absorbance) × 100%.

3. RESULTS AND DISCUSSION 3.1. Design of Chol-PEG-PpIX. Many PS molecules with extensive polycyclic aromatic structure, such as phenothiazinium, fullerenes, and xanthenes, are difficult to

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be degraded in human body and may cause severe side effects.4 In comparison, porphyrin-based PS molecules have excellent biocompatibility and good therapeutic efficacy.61−63 Specifically, protoporphyrin naturally occurs in human hemoglobin, which reduces the possibilities of systemic toxicity as well as allergic reaction.4 In our design, protoporphyrin-derived PpIX was selected as the antibacterial PS. However, anionic PpIX can only photoinactivate Gram-positive bacteria with limited efficiency.64 Herein, our aim is to create a PpIX-based PDT agent against both Gram-positive and Gram-negative bacteria. It has been reported that PpIX shows high affinity to lipid membranes,65 which is suitable for hydrophobic membrane binding. On the other hand, cholesterol with excellent membrane anchoring ability has been widely applied in cell surface modification.17−21,66 We therefore expect that the introduction of PpIX and/or cholesterol to a molecule may realize the successful binding of the molecule to the outer membrane of Gram-negative bacteria. In addition, a hydrophilic PEG chain of the molecule is also very important since it helps to improve the solubility and stability of the final molecule. We envisioned that the combination of PpIX, cholesterol, and PEG into a single molecule (i.e., Chol-PEG-PpIX) could achieve desirable cell surface binding and antibacterial performance. Besides, since all the three components have no positively charged group, the electrostatic interaction between Chol-PEG-PpIX and the bacterial surface would not dominate. In contrast, the hydrophobic binding interaction between cholesterol/PpIX and bacterial cell surface plays a key role in the bacterial surface binding of the molecule.

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3.2. Self-Assembly of Chol-PEG-PpIX in Aqueous Solution. Chol-PEG-PpIX is amphiphilic due to the presence of a hydrophilic PEG chain as well as two hydrophobic PpIX and cholesterol moieties. Therefore, the Chol-PEG-PpIX is highly water-soluble and can self-assemble into micelle-like nanoparticles in aqueous solutions. The transmission electron microscopy (TEM) image revealed that the Chol-PEG-PpIX nanoparticles are approximately 15 nm in diameter (Figure 1a), in good consistency with its hydrodynamic size (16.7 ± 3.5 nm) measured by dynamic light

scattering

(DLS)

(Figure

1b).

For comparison,

methoxyl-PEG-PpIX

(OMe-PEG-PpIX) without the cholesterol moiety and free PpIX were set as control groups. In aqueous solutions, free PpIX molecules were poorly dispersed and easily formed large aggregates (~1 µm) due to the strong π–π stacking between themselves. Similar to Chol-PEG-PpIX, the amphiphilic OMe-PEG-PpIX also showed improved dispersibility and small particle size (13.6 ± 4.4 nm), demonstrating the key role of PEG segment in forming the self-assembled nanostrutures. These results indicated that both Chol-PEG-PpIX and OMe-PEG-PpIX could form stable and small nanoparticles. We also measured the UV−vis absorption spectra of free PpIX and Chol-PEG-PpIX. As displayed in Figure S1, the Soret band (around 400 nm) of PpIX in water was significantly broadened as compared to that of PpIX monomers (in DMSO), indicating the strong π–π stacking of PpIX in aqueous solutions.67 However, Chol-PEG-PpIX in water only showed a slightly broadened Soret band in comparison with PpIX monomers, which implied the decreased π–π stacking among PpIX moieties.

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3.3. Enhanced Membrane Anchoring Ability of Chol-PEG-PpIX. To assess the membrane anchoring ability of Chol-PEG-PpIX, we adopted lipid vesicles as model cell membranes of bacteria. E. coli total lipid extract which contains all the lipid components in E. coli was utilized in this experiment to mimic the bacterial outer membrane. To begin with, we compared the fluorescence intensity of Chol-PEG-PpIX in a 0.9% NaCl solution and the vesicle solution (1 mg/mL). As displayed in Figure 1c, Chol-PEG-PpIX only emitted weak fluorescence in a 0.9% NaCl solution. This is because of the severe quenching effect of PpIX in the compact cores of the micelles formed between PpIX and cholesterol moieties via hydrophobic interaction. Strikingly, Chol-PEG-PpIX showed a 6-fold increase of fluorescence intensity after incubation with the lipid vesicle solution, indicating that the quenching effect was significantly weakened. These results clearly proved that PpIX anchored into the lipid bilayer of the vesicle. Comparatively, only a moderate elevation of fluorescence intensity was observed in the “PpIX + vesicle” and “OMe-PEG-PpIX + vesicle” samples. The above results clearly demonstrated that cholesterol could promote the interaction between PpIX molecules and lipid membranes. Hence, the membrane anchoring ability of PpIX moieties in Chol-PEG-PpIX was greatly enhanced with the assistance of cholesterol. Inspired by the enhanced fluorescence of Chol-PEG-PpIX after membrane binding, we then compared the 1O2 generation of Chol-PEG-PpIX in a 0.9% NaCl solution and the lipid vesicle solution. Singlet oxygen sensor green (SOSG) was utilized as the indicator because SOSG exhibits increased fluorescence emission at 530 nm after

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oxidation by 1O2. As expected, the SOSG’s fluorescence intensity of Chol-PEG-PpIX in the vesicle solution increased sharply upon laser irradiation and was much higher than that of Chol-PEG-PpIX in a 0.9% NaCl solution (Figure 1d), indicating that 1O2 was effectively produced after the membrane binding of Chol-PEG-PpIX.

Figure 1. Characterizations of Chol-PEG-PpIX. (a) TEM image of Chol-PEG-PpIX. (b) Hydrodynamic diameters of PpIX, OMe-PEG-PpIX, and Chol-PEG-PpIX measured by DLS. (c) Fluorescence spectra of PpIX, OMe-PEG-PpIX, and Chol-PEG-PpIX in a 0.9% NaCl solution and a lipid vesicle solution. (d) 1O2 generation of Chol-PEG-PpIX in a 0.9% NaCl solution and a vesicle solution by measuring the fluorescence intensity changes of SOSG.

3.4. Chol-PEG-PpIX Can Attach to the Cell Surface of Gram-Negative Bacteria. PDT serves as an effective strategy to kill bacteria because of the highly

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cytotoxic reactive oxygen species (ROS), mainly 1O2. However, due to the short lifespan and limited migration distance of 1O2, the antibacterial efficiency of PDT is largely dependent on the localization of PSs. To kill bacteria, PSs must be in close proximity to the outer surfaces of bacterial cells, which allows the generated 1O2 to diffuse into the cytoplasmic membrane,68 or directly penetrate into the bacterial cells.8 As compared with Gram-positive bacteria, PSs are extremely difficult to penetrate into Gram-negative bacteria due to their presence of outer membranes. Hence, enhancing the interaction of PSs with the bacterial outer membrane is crucial to increase the antibacterial PDT efficacy. Considering that Gram-positive bacteria can be easily killed by a regular PDT treatment, in this study, we mainly investigated the antibacterial mechanism and efficacy of the Chol-PEG-PpIX NPs towards Gram-negative bacteria. To understand whether Chol-PEG-PpIX would show an enhanced interaction with Gram-negative bacteria, confocal microscopic experiments were conducted. We hypothesized that the fluorescence emission of PpIX would be enhanced when PpIX molecules inserted into the bacterial outer membranes, similar to the above results of model membranes (Figure 1c). E. coli was used as a representative Gram-negative bacterial strain in our experiments. Before imaging, E. coli cells were incubated with Chol-PEG-PpIX and control reagents (free PpIX and OMe-PEG-PpIX) in a 0.9% NaCl solution at 37 oC for 30 min, respectively. As displayed in Figure 2a, the confocal fluorescence images clearly showed that the cell surfaces of E. coli cells in the Chol-PEG-PpIX-treated sample group were uniformly labeled with red fluorescence, realizing wash-free fluorescence imaging for the

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bacterial outer membranes. This result clearly demonstrated that the surface of E. coli was successfully coated by the PpIX molecules. In contrast, free PpIX- and OMe-PEG-PpIX-treated bacteria showed no detectable fluorescence. Generally, two major driving forces are involved in the physical interaction between cell surface and materials: the electrostatic interaction and the hydrophobic interaction.18 Considering that the bacterial surface is negatively charged in a physiological condition, electrostatic interaction cannot explain the successful binding because the zeta potential of Chol-PEG-PpIX in a 0.9% NaCl solution was measured to be slightly negatively charged (around −2 mV). To further confirm that the interaction between E. coli cells and Chol-PEG-PpIX NPs is not electrostatic interaction, E. coli cells were incubated with Chol-PEG-PpIX NPs in deionized water, a 10 mM NaCl solution, a 0.9% NaCl solution, and a cell phosphate buffered saline (PBS) solution, respectively. Their binding performance was then evaluated by flow cytometry (Figure 2b). Through varying the ionic strength (from 150 mM in a cell PBS solution to 10 mM in a 10 mM NaCl solution to 0 mM in deionized water) and pH (from 7.4 in a cell PBS solution to 6.0−6.5 in the other solutions) of the aqueous solution, the fluorescence intensity of various treated bacteria was rarely affected, illustrating that electrostatic interaction had little contribution to the binding process. Therefore, it is reasonable to believe that the dominant mode of the interaction between Chol-PEG-PpIX and the outer membrane of E. coli is a hydrophobic interaction. According to our previous DLS data, free PpIX easily aggregated into micro-sized particles which are too large to fully interact with bacteria. Oppositely, Chol-PEG-PpIX nanoparticles with a

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smaller size (~32 nm) have more contact areas with the cell surface of E. coli. Moreover, OMe-PEG-PpIX also failed to stain the bacterial surface, confirming the necessity to have the cholesterol moiety in the molecule. To study the role cholesterol played in the bacterial staining, cholesterol-PEG2000-fluorescein isothiocyanate (cholesterol-PEG-FITC) was chosen to incubate with E. coli cells. However, no fluorescence signal was observed for bacteria after washing treatment (Figure S2, Supporting Information), indicating that cholesterol itself could not help the whole molecule to realize the successful bacterial cell surface anchoring. The poor incorporation of cholesterol into the bacterial outer membranes may be ascribed to the higher viscosity of the lipid domain in the outer membrane and the tighter binding of phospholipids to other lipid components.57 Taken together, we propose that PpIX and cholesterol moieties in Chol-PEG-PpIX might hydrophobically anchor to the bacterial outer membranes in a synergistic manner, because both components were indispensable for the successful binding. To further verify that the PpIX moiety in Chol-PEG-PpIX indeed anchored to the bacterial outer membrane, we measured the fluorescence intensity of the nanoagent after incubating Chol-PEG-PpIX with the bacterial cells. We believe that if PpIX did not anchor to the outer membrane or only simply accumulated on the bacterial surface, the fluorescence intensity of nanoagent–bacteria mixture would exhibit no difference with that of the nanoagent alone. However, the result clearly showed that after incubating with bacteria for 30 min, the fluorescence intensity of Chol-PEG-PpIX enhanced to almost 3-fold as compared to that of non-treatment sample group (Figure

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2c). It should be noted that the autofluorescence of bacteria had been already subtracted in the spectrum. Such observation strongly demonstrated that the PpIX moiety of Chol-PEG-PpIX could insert into the bacterial outer membranes and realize fluorescence imaging of bacterial surfaces.

Figure 2. Membrane-binding study of Chol-PEG-PpIX. (a) Confocal images of E. coli cells incubated with PpIX, OMe-PEG-PpIX, and Chol-PEG-PpIX, respectively. Scale bar: 10 µm. (b) Flow cytometry analyses of E. coli cells before (control) and after treatment with Chol-PEG-PpIX NPs in deionized water, a 10 mM NaCl solution, a 0.9% NaCl solution, and a cell PBS solution, respectively. (c) Fluorescence emission spectra of Chol-PEG-PpIX before and after incubation with E. coli in 0.9% NaCl solutions. 19



3.5. Chol-PEG-PpIX Showed Enhanced Photosensitizing Efficiency. According to the above model membrane results, the increased fluorescence intensity of PpIX was well correlated with the enhanced 1O2 generation. Thus, encouraged by the excellent outer membrane-binding ability of Chol-PEG-PpIX, we studied the antibacterial activity of Chol-PEG-PpIX against E. coli upon white light irradiation. Before the experiment, the emission spectrum of the white light source was measured by a spectroradiometer to ensure that Chol-PEG-PpIX could be excited (Figure S3, Supporting Information). Colony counting demonstrated that Chol-PEG-PpIX efficiently photoinactivated the bacteria, while it exhibited no antibacterial effect in the dark (Figure 3a). To carefully evaluate its antibacterial efficacy, E. coli bacteria were first incubated with Chol-PEG-PpIX at different concentrations (0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 µM) in 0.9% NaCl solutions for 30 min and then illuminated by white light at the power density of 5 mW/cm2 for 30 min, followed by the surface plating method. As shown in Figure 3b, as low as 1 µM Chol-PEG-PpIX could effectively inactivate 99% E. coli under light irradiation and over 99.99% bacteria were killed in the presence of 16 µM Chol-PEG-PpIX. The minimal bactericidal concentration (MBC) of Chol-PEG-PpIX was determined to be 4 µM. In contrast, neither free PpIX nor OMe-PEG-PpIX exhibited any noticeable PDT effect even at a considerably high concentration of 128 µM. These results clearly demonstrated our previous hypothesis that Chol-PEG-PpIX with excellent outer membrane-binding ability could effectively inactivate E. coli upon light irradiation. Because of the

20


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cholesterol-assisted hydrophobic insertion of PpIX into the bacterial outer membrane, the antibacterial efficacy of the PDT nanoagent was remarkably enhanced as compared to that of free PpIX. The highly enhanced antibacterial efficacy of Chol-PEG-PpIX is likely caused by the following reasons. (1) With the assistance of cholesterol, the PpIX moiety can effectively insert into the lipid bilayer of bacterial outer membrane. Upon membrane binding, the quenching effect of PpIX molecules was greatly weakened. Under light irradiation, the massively produced 1O2 can directly oxidize neighboring lipid molecules and other biomolecules, causing disruption of the bacterial outer membrane. (2) It has been shown that nanoagents can easily enter bacterial cells after their membranes are damaged.69 Thus, the loss of bacterial outer membrane integrity may facilitate Chol-PEG-PpIX to penetrate across the bacterial cell wall and bind to the inner cytoplasmic cell membrane, which further promotes the death of bacteria upon light irradiation. To confirm the damage of bacterial cell surface, we examined the morphological changes of bacterial surface by scanning electron microscopy (SEM). After light irradiation, the bacteria were harvested and immediately fixed in 2.5% glutaraldehyde solutions to preserve the shape of membrane structures. SEM images showed that the surface of E. coli bacteria treated with Chol-PEG-PpIX was ruptured and exhibited many membrane defects in comparison with that of control group (Figure 3c). The result intuitively confirmed that the PDT agent could destroy the integrity of bacterial outer membrane upon light irradiation. Moreover, confocal microscopy was used to observe the drug-treated bacterial cells before and after light

21



irradiation. Interestingly, we found that the fluorescence on bacterial surfaces was brighter than that of non-irradiated group (Figure 3d). The fluorescence emission intensity of the bacterial surfaces was further quantified by the confocal image analysis software, which showed an approximately 6-fold increase of fluorescence intensity after irradiation (Figure 3e). Such observation suggested that due to the compromised integrity of bacterial outer membrane, some Chol-PEG-PpIX nanoagents might penetrate across the cell wall and accumulate on the inner cytoplasmic cell membrane. Considering that the cytoplasmic membrane, containing approximately twice as much phospholipids as the outer membrane,70 is more favorable for cholesterol binding,57 the reinforced interaction between the cytoplasmic membrane and Chol-PEG-PpIX could contribute to the increased fluorescence. In addition, we also assessed the PDT efficiency against S. aureus, a representative Gram-positive bacterial strain. As expected, colony counting results showed that Chol-PEG-PpIX NPs also enabled effective photoinactivation against S. aureus (Figure S4a, Supporting Information). We also evaluated the antibacterial activity of Chol-PEG-PpIX NPs in a concentration-dependent manner (Figure S4b, Supporting Information). The MBC of the nanoagent against S. aureus was tested to be 0.5 µM, confirming the intrinsic susceptibility of Gram-positive bacteria towards PDT. Moreover, only 2 µM Chol-PEG-PpIX NPs achieved an excellent antibacterial efficacy upon light irradiation with over 99.99% S. aureus cells being killed. Therefore, Chol-PEG-PpIX can inactivate both Gram-negative and Gram-positive bacteria at very low concentrations upon mild light irradiation, which may be

22


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potentially used as a broad-spectrum antibacterial agent.

Figure 3. Evaluation of the antibacterial efficacy of Chol-PEG-PpIX against E. coli after light irradiation. (a) Agar plate photographs for E. coli before (control) and after treatment with light, Chol-PEG-PpIX, and Chol-PEG-PpIX + light, respectively. (b) Dependence of bacterial survival fraction on the concentration of PpIX, OMe-PEG-PpIX, and Chol-PEG-PpIX under light irradiation. (c) SEM images of E. coli cells before (control) and after treatment with Chol-PEG-PpIX and light. Scale bar = 1 µm. (d) Confocal fluorescence images of

23



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Chol-PEG-PpIX-treated E. coli cells before and after light irradiation. (e) Fluorescence (FL) intensity of the surfaces of Chol-PEG-PpIX-treated E. coli cells before and after light irradiation. The FL intensity was quantified by the confocal image analysis software.

3.6.

Cytotoxicity

and

Hemocompatibility

of

Chol-PEG-PpIX.

The

abovementioned results demonstrate that Chol-PEG-PpIX can serve as effective antibacterial nanoagents. Next, we evaluated the cytotoxicity of Chol-PEG-PpIX towards L02 cells (human normal hepatocyte). As presented in Figure 4a, the cell viability exhibited only a slight decrease with increasing Chol-PEG-PpIX concentration. The Chol-PEG-PpIX showed negligible cytotoxicity at a concentration of 30 µM, which is already much higher than the MIC of Chol-PEG-PpIX for E. coli. In addition, the hemolysis assay showed that Chol-PEG-PpIX had negligible hemolytic activity towards red blood cells (RBCs) even at the highest concentration of 50 µM (Figure 4b), confirming the good hemocompatibility of Chol-PEG-PpIX. Therefore, the nanoagent possesses excellent biocompatibility with mammalian cells including RBCs.

Figure 4. Biocompatibility evaluation of Chol-PEG-PpIX. (a) Viabilities of L02 cells after being treated with different concentrations of Chol-PEG-PpIX. (b) Hemolysis rates of RBCs

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after incubation with various concentrations of Chol-PEG-PpIX. H2O and PBS were set as the positive control and negative control, respectively.

4. CONCLUSIONS In summary, we developed a bacterial surface targeting PDT nanoagent composed of Chol-PEG-PpIX

that

could

indiscriminately

kill

both

Gram-negative

and

Gram-positive bacteria. The Chol-PEG-PpIX compound can be readily synthesized through

the

one-step

chemical

conjugation

of

two

commercial

reagents

(Cholesterol-PEG2000-NH2 and PpIX). When incubating with Gram-negative bacteria, the self-assembled Chol-PEG-PpIX NPs would dis-assemble and the cholesterol moieties could significantly facilitate the hydrophobic anchoring of PpIX on the bacterial outer membrane. The membrane-bound PpIX molecules with weakened self-quenching effect exhibited significantly increased fluorescence emission as well as 1O2 generation. The highly reactive 1O2 could cause the peroxidation of membrane lipids, resulting in the severely damaged bacterial cell surface. The enhanced permeability of the bacterial outer membrane further ensured the nanoagent to cross the cell wall and bind to the inner cytoplasmic membrane, which promoted the photodynamic killing of bacteria. Besides, the nanoagent is equally effective against Gram-positive bacteria. Therefore, such a hydrophobic interaction-based bacterial cell surface engineering strategy can successfully realize bacterial cell surface imaging-guided photo-inactivation of both Gram-negative and Gram-positive bacteria. To the best of our knowledge, the present work represents the first example to construct a fully hydrophobic interaction-based and outer membrane-anchored antibacterial PDT nanoagent, which may shed new light on the design of next-generation photosensitizers for photodynamic inactivation of

25



pathogenic bacteria.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Confocal fluorescence images of E. coli stained by Chol-PEG-FITC and PDT results of Chol-PEG-PpIX NPs against S. aureus (PDF)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected], [email protected]. Author Contributions §

H.R.J. and Y.X.Z. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (21673037), Fundamental Research Funds for the Central Universities (2242015R30016), Six Talents Peak Project in Jiangsu Province (2015-SWYY-003), and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. ZC acknowledges the support from the University of

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Michigan.

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Cholesterol-Assisted Bacterial Cell Surface Engineering for

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