Self-Assembled Rose Bengal-Exopolysaccharide Nanoparticles for

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Functional Nanostructured Materials (including low-D carbon)

Self-assembled Rose Bengal-Exopolysaccharide Nanoparticles for Improved Photodynamic Inactivation of Bacteria by Enhancing Singlet Oxygen Generation Directly in the Solution Chengcheng Li, Fengming Lin, Wei Sun, Fu-Gen Wu, Hang Yang, Roujing Lv, Ya-Xuan Zhu, Hao-Ran Jia, Chu Wang, Ge Gao, and Zhan Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01545 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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ACS Applied Materials & Interfaces

Self-assembled

Rose

Nanoparticles

for

Bengal-Exopolysaccharide Improved

Photodynamic

Inactivation of Bacteria by Enhancing Singlet Oxygen Generation Directly in the Solution Chengcheng Li,a Fengming Lin,a* Wei Sun,a Fu-Gen Wu,a Hang Yang,a Roujing Lv,a Ya-Xuan Zhu,a Hao-Ran Jia,a Chu Wang,a Ge Gao,a Zhan Chenb* a

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

Engineering, Southeast University, Nanjing 210096, China b

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

Ann Arbor, MI 48109, United States

Abstract: It is of great value to develop new antibacterial photodynamic therapy (PDT)

strategies

to

improve

antibacterial

PDT

efficacy

of

non-cationic

photosensitizers without introducing cytotoxicvdfity that is a great challenge for current leading efforts on antimicrobial PDT based on cell surface engineering. In this research, the hydrophobic and anionic photosensitizer rose bengal (RB) was chemically conjugated with bacterial exopolysaccharide (EPS) to generate an amphiphilic and negatively-charged compound EPS-RB that could self-assemble into nanoparticles (NPs) in solution. These EPS-RB NPs possessed an increased singlet oxygen generation property in solution. As a result, EPS-RB exhibited improved photoinactivation for both Gram-negative and Gram-positive bacteria, leading to a record low RB working concentration, 8 µM or 500 nM for E. coli or S. aureus. Upon 1

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light irradiation, more EPS-RB bound to cell surface and penetrated into bacteria than RB, with EPS-RB staying around the cell surface of most irradiated E. coli while entering all of irradiated S. aureus. Both scanning electron microscopy and fluorescence confocal imaging results show that the cell membrane of E. coli was damaged heavily, but not S. aureus. All these observations indicate that both the enhanced singlet oxygen production of EPS-RB NPs in solution and their consequently increased membrane binding and cellular penetration into the bacteria through the damaged cell membrane, contribute to its significantly improved bacterial photoinactivation efficiency. In addition, EPS-RB has low cytotoxicity and negligible hemolytic activity, showing great biocompatibility. Therefore, the construction of EPS-RB provides a new strategy for the PDT effectiveness improvement of the separated cell-sensitizer systems, and thus the design of next generation antimicrobial agents. Keywords: rose bengal, antibacterial, photodynamic therapy, exopolysaccharide, singlet oxygen Introduction Photodynamic therapy (PDT) presents an effective strategy for anti-cancer1 and anti-bacteria.2, 3 It employs light to sensitize drugs/dyes (called photosensitizers, PSs) in the presence of oxygen to generate reactive oxygen species (ROSs), mainly singlet oxygen together with free radicals, which causes cell apoptosis/necrosis and tissue destruction.4 Particularly, antimicrobial PDT is considered to be a promising alternative way to conventional antibiotics to eradicate the regular and drug-resistant 2


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(DR) bacteria.3, 5 DR bacteria have been reported to be as susceptible as non-DR bacteria to PDT,3, 6 making PDT promising for combating bacteria drug-resistance. Furthermore, it is believed that PDT would not easily generate antibiotic-resistance7 when attacking microorganisms by using ROSs to irreversibly damage the important biomolecules,8 such as DNAs,9 proteins,10 and lipids.11 PDT only directly affects molecules and structures that are proximal to PSs, due to the high reactivity and short half-life of singlet oxygen and hydroxyl radicals. The half-life of singlet oxygen is < 0.04 µs and consequently its action radius is < 0.02 µm in biological system.12 Therefore, the PSs’ localization largely determines the antibacterial efficiency of PDT. It is far preferred that PSs anchor to cell membrane/wall or penetrate into the cells. However, most PSs are hydrophobic and have poor water-solubility, preventing them from targeting and entering cells.13 To improve their water-dispersability and cellular uptake, PSs are commonly physically encapsulated in or chemically conjugated with nanocarriers such as metal nanoparticles (NPs),14 polymeric NPs,15-17 upconversion NPs,18, 19 micelles,20 liposomes20, 21 and so on. These efforts are commonly based on cell surface engineering via electrostatic interaction,22, 23 hydrophobic interaction24 and covalent conjugation, which often have disadvantages such as high cytotoxicity, low biocompatibility, complicated modification methods, and limited effectiveness. Another drawback of PDT that is urgently needed to resolve is its less antibacterial efficacy on Gram-negative bacteria than on Gram-positive ones.25-27 Compared to Gram-positive bacteria and animal cells, the Gram-negative bacteria have an additional layer in the cell wall structure, the outer membrane, serving as an extra 3



effective penetration barrier to many drugs including PSs.25-28 Strategies have been developed to increase the PDT efficacy against Gram-negative bacteria. As a straightforward way, the cell membrane disrupting agents such as toluene29 and the metal ion chelator EDTA30, 31 were implemented with PSs to pretreat Gram-negative bacteria, making the outer membrane more permeable to PSs and enhancing the effectiveness of PDT in killing Gram-negative bacteria. Another approach to overcome the initial difficulty in photodynamic inactivation on Gram-negative bacteria, is to utilize PSs with an intrinsic positive charge like methylene blue,26 toluidine blue O26 and conjugated polyelectrolytes,32 or couple PSs with the positively charged compounds such as polymixin B,33 poly-L-lysine,34 and polyethylenimine.35 Through electrostatic interaction, these cationic agents effectively interact with the negatively charged lipopolysaccharides, a rich component of the outer membrane of Gram-negative bacteria, which efficiently enables the photoinactivation of Gram-negative bacteria. Nevertheless, these strategies have concerned cytotoxicity and non-biocompatibility issues. For example, EDTA inhibits mammalian cell growth36 and might result in tissue inflammation,36 while most cationic agents are toxic to mammalian cells, particularly red blood cells, and might trigger immunogenic reactions.37 Thus, it is worth developing new antibacterial PDT strategies toward Gram-negative bacteria that are not based on either cell membrane disrupting or electrostatic interaction. Rose bengal (RB), a hydrophobic and anionic xanthene dye,1, 27, 31 is well-known as an anionic photosensitizer used in PDT. RB produces nearly 76% singlet oxygen 4


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under 532 nm light irradiation.1 RB showed high photoinactivation action on Gram-positive bacteria and some fungi, but not on Gram-negative bacteria.31 Moreover, it suffers from poor cellular uptake, limiting its effective application in antibacterial and anti-cancer PDT.38 Therefore, studies have been carried out on the combination of RB with cationic carriers, such as a cationic phosphorus dendrimer,39 polycationic chitosan,40 and positively charged peptide

41, 42

to increase the positive

charge on the surface, realizing a better binding with and penetration through the bacterial cell membrane that was negatively charged. This causes cytotoxicity as we mentioned above. In this work, RB was chemically conjugated with bacterial exopolysaccharide (EPS) to generate an amphiphilic compound EPS-RB. EPS-RB can self-assemble into nanoparticles (NPs) in solution with increased singlet oxygen generation. More importantly,

EPS-RB

could

display

improved

photoinactivation

for

both

Gram-negative and Gram-positive bacteria. After light irradiation, more EPS-RB enters bacteria than RB. The effect of EPS-RB on the bacterial morphology and its biocompatibility were also investigated. Scheme 1. Schematic Illustration Showing Photodynamic Inactivation of Bacteria by EPS-RB.

5



Materials and methods 2.1 Materials EPS-605 was purified from the fermentation broth of Lactobacillus plantarum LCC-605, as described in our previous study.10 1-Ethyl-3-(3-(dimethylamino) propyl) carbodiimide hydrochloride (EDC · HCl) was purchased from Sigma-Aldrich (St. Louis, MO, USA). N-hydroxysuccinimide (NHS), and dimethyl sulfoxide (DMSO) were obtained from Aladdin Chemistry Co., Ltd (Shanghai, China). Deionized water was from a Milli-Q synthesis system (Billerica，MA，USA). Singlet oxygen sensor green (SOSG) kit was acquired from Molecular Probes Inc. (Eugene, Oregon, USA). Dialysis bags with the molecular weight cutoff (MWCO) of 10 kDa were ordered from Sangon Biotech. Co., Ltd. (Shanghai, China). All other chemicals used in this study were of analytical grade. 2.2 Synthesis of EPS-RB 3.1 mg RB, 8.0 mg EDC•HCl, and 4.8 mg NHS were dissolved in 9 mL DMSO, mixed 6


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and incubated for 1 h at room temperature with 90 rpm, followed by the addition of 1 mL EPS-605 (0.8 mg/mL) and further reacted overnight. The solution was dialyzed (MWCO = 10 kDa) against DMSO for 3 days and against H2O for 1 day successively. Finally, the as-prepared EPS-RB was lyophilized and stored in dark at −20 °C for future use. 2.3 Characterization of EPS-RB Transmission electron microscopy (TEM) was employed to determine the morphology and size of EPS-RB in aqueous solution. Typically, a drop of EPS-RB (100 µM) in water was deposited on a 400-mesh carbon-coated copper grid, dried at room temperature, and examined using a transmission electron microscope (JEM-2100; JEOL Ltd., Japan). The size distribution was analyzed by the Nano Measure software 1.2. The UV-vis absorption spectra of free RB and EPS-RB were obtained with a Shimadzu UV-2600 spectrophotometer. Size distribution of EPS-RB in 0.9% NaCl was performed using dynamic light scattering (DLS) with a Zetasizer 3000 instrument (Malvern Instruments, Nano ZS, United Kingdom). 2.4 1O2 Generation assay The singlet oxygen (1O2) generation was determined using the singlet oxygen sensor green (SOSG) kit. Briefly, 1 µL of SOSG dissolved in methanol was mixed with 2 mL different samples as indicated in the text below and irradiated by a laser (532 nm) at a power density of 14 mW/cm2. Then, the fluorescence intensity of the solution was recorded at 530 nm every 1 minute until equilibration was reached. 2.5 Fluorescence intensity measurement 7



EPS-RB or RB was mixed with 1 mL E. coli culture (OD600 = 0.5) to reach a final concentration of 10 µM and incubated at 37 oC for 30 min. Then, the fluorescence intensity of the samples was measured at 552 nm by a spectrofluorophotometer (RF-5301PC, Shimadzu, Japan). 2.6 Photodynamic inactivation of bacteria E. coli and S. aureus were cultivated in lysogeny broth (LB) medium at 37 oC with 180 rpm. The initial optical density of the bacteria was determined at 600 nm (OD600) via a UV-vis spectrophotoscopymeter. The overnight culture of E. coli or S. aureus was washed twice with 0.9% NaCl solution and then diluted to OD600 = 0.5, which was subsequently resuspended in 0.9% NaCl solution. 10 µL bacterial suspensions were mixed with 90 µL of EPS-RB, RB or EPS in 0.9% NaCl solution at desired concentrations. The final RB concentrations of 2, 4, 5, 5.5, 6, 7, 8 and 16 µM were tested, while the final EPS concentrations tested included 0.16, 0.32, 0.4, 0.44, 0.48, 0.56, 0.64 and 1.3 mg/mL. After incubation at 37 oC for 30 min, the treated bacteria were irradiated by a white light (5 mW/cm2) for 10 min. Then, 100 µL of properly diluted bacterial solutions were plated on LB agar plates, and the number of bacterial colonies was counted after incubation for 24 h at 37 oC. The power density of white light was measured according to the method in the literature.24 2.7 Confocal image and scanning electron microscopy The bacterial cells before and after light irradiation as described in section 2.6 were washed with 0.9% NaCl solution, and examined under confocal laser scanning microscope TCS SP8 (Leica, Germany) with a 100 × oil immersion objective. The 8


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excitation wavelength was 552 nm and the fluorescence emission was detected in the wavelength range of 582-670 nm. Meanwhile, scanning electron microscopy (SEM) was utilized to monitor the morphological changes of bacterial surface using an FESEM (Zeiss Ultra Plus, Germany). The samples were prepared as following: After the light irradiation in the presence of 10 µM EPS-RB, E. coli was washed three times using 0.9% NaCl solution and fixed with 4% glutaraldehyde solution for 24 h to preserve its original shape. After being dehydrated with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol, a drop of bacteria in 100% ethanol was deposited on a silicon slide for SEM examination. The non-treated E. coli was set as the control. 2.8 MTT assay ATII cells, normal human lung cells, were cultured in Dulbecco's modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) and 100 IU/mL penicillin-streptomycin at 37 oC with 5% CO2. ATII 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 RB, EPS and EPS-RB respectively at different concentrations (5, 10, 20, and 25 µM) for 24 h at 37 oC with 5% CO2. 10 µL MTT (5 mg/mL) was added into each well. After incubation for another 4 h, the solution was discarded and 150 µL DMSO was added. Then the absorbance was recorded at 450 nm using a microplate reader (Thermo-Scientific, Multiskan FC, USA). 2.9 Hemolysis assay Red blood cells (RBCs) were isolated from the whole mouse blood by centrifugation at 2500 rpm for 5 min, and resuspended in 0.9% NaCl solution. The collected RBCs 9



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were treated with EPS-RB or RB with the final RB concentrations of 1, 5, 10, 20, and 50 µM, respectively. After incubation at 37 °C for 1 h, the mixture was centrifuged at 4000 rpm for 5 min. RBCs incubated with PBS and ultrapure water were carried out as the negative control and the positive control, respectively. The percentage of hemolysis was calculated using the following equation: Hemolysis % = (sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) x100%. 3. Results and discussion 3.1 EPS-RB self-assembled into nanoparticles with enhanced singlet oxygen generation Microbial exopolysaccharide EPS-605 purified from L. plantarum LCC-605

10

in

our lab contains -NH2 and -COOH groups as revealed by Fourier transform infrared (FTIR)and X-ray photoelectron spectroscopy (XPS) spectra, enabling further modification by chemical conjugation. It is amphiphilic, negatively charged, and water soluble.10 We thus envision that EPS-605 could be a good carrier for PSs. Therefore, EPS-605 was chemically coupled with hydrophobic and anionic RB by the EDC/NHS coupling method using the carboxylic groups (-COOH) of RB and the amine groups (-NH2) of EPS-605. The TEM image of the as-synthesized EPS-RB in 0.9% NaCl revealed that EPS-RB can self-assemble into nanoparticles with an average diameter of 35.2 nm (Figure 1a), of which the hydrodynamic diameter measured by DLS was 82.8 nm (Figure S1). In water, EPS-RB displays a nearly identical UV-vis absorption spectrum to RB (Figure 1b), showing a narrow peak at 10


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550 nm. EPS-RB was negatively charged with a zeta potential of -12.6 mV in 0.9% NaCl (Figure 1c), which is reasonable because the two raw materials RB and EPS-605, were also negatively charged (Figure 1c). To evaluate the physiological stability of the synthesized NPs in the medium, we have measured both the size and the zeta potential of EPS-RB in 0.9% NaCl at 0, 24 and 48 h (Figure S5), which remained constant within 48 h. No aggregation was observed within 48 h. These demonstrated that the NPs were stable in solution within 48 h. Interestingly, enhanced singlet oxygen generation was observed for EPS-RB compared to free RB under light irradiation. The fluorescence intensity of SOSG at 530 nm, which is increased when SOSG is oxidized by 1O2, was implemented as the indicator of the 1O2 abundance in the solutions (Figure 1d and Figure S2). EPS-RB generates more 1O2 than free RB upon white light irradiation, which was further enhanced along with the increase of the irradiation time. At 10 min when the 1O2 production of both EPS-RB and RB reached a plateau, the 1O2 production of EPS-RB was increased by 27% compared to that of RB. This increased production of 1O2 should be beneficial for antibacterial PDT of EPS-RB. Meanwhile, the fluorescence intensity of EPS-RB was 28% higher than that of RB (Figure 1e). This higher singlet oxygen generation and stronger fluorescence emission may be due to the highly viscous environment inside the EPS-RB nanoparticles produced by EPS-605. In this highly viscous context, the rotation and intermolecular collision of RB molecular would be reduced and hence their π-π interaction would be weakened, resulting in less aggregates and lower self-quenching of EPS-RB under light irradiation, as compared 11



to free RB in the less viscous water. 43 A similar pattern of PSs was also observed when they were loaded to injectable hydrogels with high viscosity, which has been successfully applied in imaging-guided photodynamic cancer therapy.44

Figure 1. Characterization of EPS-RB. (a) TEM image of EPS-RB and the corresponding size statistical analysis. (b) UV−vis absorption spectra of RB and EPS-RB in water. (c) Zeta potentials of RB, EPS and EPS-RB in 0.9% NaCl. (d) 1O2 generation of RB, EPS and EPS-RB in 0.9% NaCl as measured by the fluorescence intensity changes of SOSG at 530 nm with the excitation wavelength of 504 nm. (e) Fluorescence emission spectra of RB and EPS-RB in 0.9% NaCl solution. The excitation wavelength was 552 nm.

3.2 EPS-RB

improved photoinactivation for both Gram-negative and

Gram-positive bacteria 12


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Inspired by the improved 1O2 production of EPS-RB, we measured the antimicrobial PDT ability of EPS-RB against E. coli and S. aureus that represent the model Gram-negative and Gram-positive bacteria, respectively. E. coli was incubated with different concentrations (2, 4, 5, 5.5, 6, 7, 8 and 16 µM) of RB or EPS-RB in 0.9% NaCl for 30 min and irradiated by white light (5 mW/cm2), followed by the standard plate count method. The irradiation time was set to 10 min when the 1O2 production of both EPS-RB and RB reached a plateau. Without light irradiation, no antibacterial effect was observed for RB, EPS or EPS-RB as well as EPS under light irradiation (Figure 2b, 2d and Figure S3). Upon white light irradiation, it required 5.5 µM, 7 µM and 8 µM EPS-RB to annihilate 99%, 99.99% and 100% E. coli, respectively, representing 83%, 70% and 50% of that of free RB (7.5 µM, 12 µM and 16 µM) (Figure 2b). In terms of S. aureus, only 5 nM, 50 nM and 500 nM EPS-RB was needed to eradicate 99%, 99.99% and 100% cells, much less than that of free RB (10 nM, 250 nM and 1000 nM) (Figure 2d).

13



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Figure 2. Evaluation of the photodynamic inactivation of EPS-RB against E. coli (a and b) and S. aureus (c and d). Agar plate photographs of E. coli (a) and S. aureus (c) treated with RB or EPS-RB respectively were taken under (non-) light irradiation conditions. The corresponding dependence of bacterial survival fraction on the concentration of RB and EPS-RB was measured (b and d). The circle represents there is no bacteria survival. The error bars indicate the standard deviation of three independent replicates.

RB has been conjugated to silica beads,45 core-shell silver-silica nanoparticles

46

and chitosan nanoparticles47 to improve its photoinactivation of Gram-positive bacteria, reducing its working concentration to1.5-14 µM.45, 47, 48 Herein, only 500 nM 14


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EPS-RB was enough to completely inactivate Gram-positive bacteria, lower than any reported working concentrations for photoinactivation of Gram-positive bacteria using RB in the literature. Only core-shell silver-silica nanoparticles had been explored to overcome the photoinactiaction ineffectiveness of RB on Gram-negative bacteria, in which the working concentration of RB is 13 µM,46 higher than 8 µM achieved by EPS-RB in this work. Clearly, EPS-RB exhibit a much better antimicrobial PDT ability toward both Gram-negative and Gram-positive bacteria than that of RB. It is worth noting that only 10-minute irradiation was used in our study, much shorter than the irradiation time 30-60 min frequently utilized in literature.24, 45, 49 The impact of the irradiation time on bacterial photoinactivation of EPS-RB was investigated using E. coli. The photoinactivation efficacy of EPS-RB at a fixed concentration of 5.5 µM was improved remarkably along with increased irradiation time from 5 min to 60 min. 5.5 µM EPS-RB inactivated 99% E. coli after 10-minute irradiation, which was improved to 100 % by increasing the irradiation time to 30 min (Figure 3). When using 10-minute irradiation, 8 µM EPS-RB was required to kill 100 % E. coli (Figure 2a). The working concentration of EPS-RB reduced along with the increasing irradiation time. This illustrates that the enhancement of bacterial photoinactivation by EPS-RB can be increased with a longer irradiation time. The photoinactivation efficacy enhancement of RB by increasing the irradiation time was far less than that of EPS-RB.

15



Figure 3. Evaluation of the irradiation time on the effectiveness of EPS-RB against E. coli. The concentration of EPS-RB was 5.5 µM and the power density was 5 mW/cm2. The circle represents there is no bacteria survival. The error bars indicate the standard deviation of three independent replicates.

3.3 More EPS-RB NPs entered bacteria than RB after light irradiation Fluorescence confocal microscopy was utilized to track RB in the drug-treated bacterial cells before and after light irradiation, given that RB displays red fluorescence. Interestingly, we found that with the treatment of EPS-RB or RB the fluorescence of E. coli was much brighter after irradiation in comparison with that before irradiation (Figure 4a). After irradiation, E. coli treated with EPS-RB displayed markedly stronger fluorescence than that treated with RB. A similar pattern was observed for S. aureus (Figure 4b). These results demonstrated that more EPS-RB than RB could be anchored to the cell membrane/wall and penetrated across the cell wall and accumulated inside the cells as well, regardless of the Gram-type, leading to 16


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a fascinating photodynamic inactivation of both Gram-negative and Gram-positive bacteria.

Figure 4. Tracking RB in the drug-treated bacterial cells before and after light irradiation. Confocal images of E. coli (a) and S. aureus (b) with RB and EPS-RB under (non-) irradiation conditions. The concentrations of RB for E. coli and S. aureus were 10 µM and 1 µM, respectively. All the confocal images share a same scale bar of 20 µm. The area marked with the white square in (a) and (b) was enlarged and presented in (c) and (d), respectively.

3.4 EPS-RB damaged the cell membrane of E. coli, but not S. aureus To monitor the effect of EPS-RB on the morphology of bacterial surface, SEM experiments were performed on the irradiated bacteria. Compared to the untreated group that showed a normal rod shape with smooth surface, E. coli treated with 17



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EPS-RB after irradiation displayed wrinkled abnormalities with clefts distributed around the cell surface, indicating a disruption of the membrane integrity (Figure 5a). However, no substantial morphology change was observed in the SEM picture of the irradiated S. aureus (Figure 5a). These findings were further evidenced by the fluorescence confocal image results. The cell membrane dye GC-Chol-PEG-FITC with green fluorescence developed in our lab can be used to indicate the integrity of cell membrane

22

and track the RB

distribution. If the cell membrane was not damaged, GC-Chol-PEG-FITC could selectively bind to the cell membrane without falling off or cellular internalization, featuring

strong

fluorescence

in

the

cell

membrane;

Otherwise,

the

GC-Chol-PEG-FITC would fall off the cell membrane or enter the cells, displaying weak fluorescence or even no fluorescence on the cell membrane.22 When co-staining the irradiated E. coli with GC-Chol-PEG-FITC, only weak green fluorescence was observed in the cell membrane of most cells, and strong green fluorescence was found for the whole body of only a small part of cells which was overlapped well with the red fluorescence of EPS-RB, generating yellow fluorescence (Figure 5b). This suggests the irradiated E. coli had varied degrees of cell membrane damage. EPS-RB NPs were around or bound to the cell surface of most E. coli, while entering a few of them. In contrast, the cell membrane of the most irradiated S. aureus exhibited strong green fluorescence with the rest ones full of strong green fluorescence, indicating that the cell membrane of the most irradiated S. aureus was not impaired significantly (Figure 5b). EPS-RB NPs were observed in the whole cells for all the irradiated S. 18


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aureus (Figure 5b). Altogether, both SEM and fluorescence confocal imaging results show that the cell membrane of E. coli was damaged heavily, but not S. aureus. Also, EPS-RB stays around/on the cell surface of most irradiated E. coli, while entering all of irradiated S. aureus. The relative porous layer of peptidoglycan surrounding the cytoplasmic membrane of Gram-positive bacteria allows the penetration of EPS-RB without destroying their cell membrane.50 However, it is dispensable to destroy the cell membrane/wall of Gram-negative bacteria to enable EPS-RB to enter them, due to their extra positively-charged outer membrane.25-28 This might well explain that the RB working concentration in the killing Gram-negative (8 µM) was much higher than that in killing Gram-positive (500 nM) bacteria (Figure 2).

19



Figure 5. (a) SEM images of E. coli and S. aureus cells before (control) and after treatment with EPS-RB under light irradiation (Drug) (scale bar = 2 µm). (b) Confocal images of E. coli and S. aureus co-stained with GC-Chol-PEG-FITC.

The possible reasons were proposed for the effectively enhanced antimicrobial PDT ability of EPS-RB. The chemical conjugation of RB with EPS enables the 20


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encapsulation of the hydrophobic RB into the amphiphilic EPS polymer, which spontaneously forms nanospheres embedded with highly viscous microenvironment, decreasing the quenching effect faced by free RB and resulting in markedly enhanced singlet oxygen production in solution. This enhancement of singlet oxygen production can more efficiently oxidize proximal lipid molecules and other biomolecules, causing substantial disruption of the bacterial outer membrane. More EPS-RB anchor to and diffuse across the disrupted bacterial cell membrane and accumulate inside the cells, because nanoparticles can easily enter bacteria with damaged cell membrane,51 further boosting the photoinactivation effect. Thus, both the enhanced singlet oxygen production of EPS-RB NPs in solution and their consequently increased membrane binding and cellular penetration into the bacteria contribute to their significantly improved bacterial photoinactivation efficiency. 3.5 EPS-RB NPs displayed good biocompatibility For their practical applications, the safety of EPS-RB NPs was assessed by cytotoxicity measurement and hemolysis assay. EPS-RB exhibited nearly no cytotoxicity toward AT II cells at 10 µM that is higher than its working concentrations for E. coli (8.0 µM) and S. aureus (500 nM) (Figure 6a). When the concentration of EPS-RB was increased up to 25 µM, only a slight decrease of the cell viability was observed with > 80% AT II cell alive (Figure 5a). The hemolytic activity of EPS-RB on red blood cells (RBCs) was also negligible. Only 2.1% hemolysis was observed with 50 µM EPS-RB, much lower than 10.3% for RB (Figure 6b), the photos of hemolysis behaviors of RBCs after incubation with various concentrations of RB, 21



EPS and EPS-RB were also taken as Figure S4. Noticeably, EPS-RB has good biocompatibility.

Figure 6. Biocompatibility evaluation of EPS-RB. (a) Viabilities of AT II cells treated with different concentrations of RB, EPS and EPS-RB. (b) Hemolysis rates of RBCs after incubation with various concentrations of RB, EPS and EPS-RB. RBCs in H2O and PBS were set as the positive and negative controls, respectively. The error bars indicate the standard deviation of three independent replicates.

Conclusion EPS-RB, fabricated by chemical conjugation of RB with bacterial exopolysaccharide EPS-605, can self-assemble into 35.2 nm nanoparticles with markedly enhanced singlet oxygen production in solution under light irradiation. Particularly, EPS-RB was found to significantly improve bacterial photoinactivation efficiency regardless of Gram types. This enhancement of singlet oxygen production upon light irradiation caused disruption of the bacterial cell membrane that led to more EPS-RB anchoring to and diffusing across the disrupted bacterial cell membrane, as revealed by SEM and 22


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fluorescence imaging results. All these facts contribute to the effectively enhanced antimicrobial PDT ability of EPS-RB. Moreover, both cytotoxicity and hemolytic activity of EPS-RB were negligible, avoiding the cytotoxicity drawback faced by many current photosensitizers based on cell surface engineering. Currently, the antimicrobial PDT efficacy was commonly improved by effective PS loading on the bacterial surface, which was achieved using cell surface engineering via electrostatic interaction,22, 23 hydrophobic interaction24 and covalent conjugation.52 Nevertheless, enhancing the photosensitizing efficiency directly in solution, as achieved by EPS-RB here, is of great value for neutral and anion PSs, but is lack of study. For these PSs, anchoring to the cell membrane/wall and penetrating into the cells are very weak before irradiation even with a long time incubation, in which case photodynamic action must initiate outside the cell surface. Therefore, the construction of EPS-RB provides a new strategy to improve the PDT effectiveness of these cell/non-cationic PS systems with excellent biocompatibility. The advantages of enhancing the singlet oxygen generation in the solution include less cytotoxicity and no generation of antibiotic resistance. AUTHOR INFORMATION Corresponding Author * E-mail: (F. M. L.) [email protected]; (Z. C.) [email protected]. Funding This work was supported by the National Natural Science Foundation of China (31700040), the Fundamental Research Funds for the Central Universities and a 23



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TOC Graphic:

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Self-Assembled Rose Bengal-Exopolysaccharide Nanoparticles for

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