PEGylated Self-Assembled Nano-Bacitracin A - ACS Publications

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Biological and Medical Applications of Materials and Interfaces

PEGylated Self-assembled Nano-bacitracin A : Probing the Antibacterial Mechanism and Real-Time Tracing of Target Delivery In Vivo Wei Hong, Yining Zhao, Yuru Guo, Chengcheng Huang, Peng Qiu, Jia Zhu, Chun Chu, Hong Shi, and Mingchun Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00135 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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ACS Applied Materials & Interfaces

PEGylated Self-assembled Nano-bacitracin A: Probing the Antibacterial Mechanism and Real-Time Tracing of Target Delivery In Vivo Wei Honga*, Yining Zhaoa, Yuru Guoa, Chengcheng Huanga, Peng Qiua, Jia Zhub, Chun Chub, Hong Shic, Mingchun Liua

a

Key Laboratory of Zoonosis of Liaoning Province, College of Animal

Science and Veterinary Medicine, Shenyang Agricultural University, Dongling Road 120, Shenyang, Liaoning Province, 110866, P.R. China b

School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road

103, Shenyang, Liaoning Province, 110016, P.R. China c

School of Pharmacy, China Pharmaceutical University, Longmian

Avenue 639, Jiangning District, Nanjing, 211198, P.R. China

a,*

Corresponding author .

Tel.: +86-24-88487156; Fax:+86-24-88487156; E-mail address: [email protected]

1

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ABSTRACT Although

nano-self-assemblies

of

hydrophobic-modified

bacitracin

A with

PLGA

(Nano-BAPLGA) have demonstrated promising antibacterial activities, the application of Nano-BAPLGA was severely compromised by low water solubility. In this study, a series of PEGylated PLGA copolymers were selected to conjugate the N-terminus of bacitracin A in order to construct PEGylated self-assembled Nano-BAs and to further develop nano-self-assemblies of bacitracin A with strong antibacterial potency and high solubility. Compared with Nano-BAPLGA, all PEGylated Nano-BAs, except Nano-BA5K, exhibited strong antibacterial efficiency against both gram-positive and gram-negative bacteria by inducing loss of cytoplasmic membrane potential, membrane permeabilization and leakage of calcein from artificial cell membranes. Mechanism studies of PEGylated Nano-BAs against gram-negative bacteria indicated that the strong hydrophobic and van der Waals interactions between PLGA and lipopolysaccharide (LPS) could bind, neutralize and disassociate LPS, facilitating cellular uptake of the nanoparticles, which could destabilize the membrane, resulting in cell death. Moreover, PEGylated Nano-BAs (Nano-BA12K) with longer PLGA block were expected to occupy a higher local density of BA mass on the surface and stronger hydrophobic and van der Waals interactions with LPS, which were responsible for the enhanced antibacterial activity against gram-positive and emerging antibacterial activity of gram-negative bacteria, respectively. In vivo imaging verified that PEGylated Nano-BAs exhibited higher inflammatory tissue distribution and longer circulation time than Nano-BAPLGA. Therefore, although PEGylation did not affect antibacterial activity, it is necessary for target delivery and resistance to clearance of the observed PEGylated Nano-BAs. In vivo, Nano-BA12K also showed the highest therapeutic index against infection burden in a mouse thigh infection model among the tested formulations, which showed good correlation with the in vitro results. In conclusion, Nano-BA12K showed high efficacy in the treatment of invasive infections. This new approach of constructing nanoantibiotics by modification of commercially available antibiotics with PEGylated copolymers is safe, cost-effective and environmentally friendly. KEYWORDS: PEGylated nano-self-assemblies, antibacterial mechanism, hydrophobic and van der Waals interactions, LPS, long circulation time, target delivery.

2


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1. INTRODUCTION Bacitracin is an important non-ribosomal polypeptide antibiotic composed of a mixture of cyclic polypeptides.1 Previous studies has shown that the component designated as A (BA) is the most microbiologically active.2 BA possesses potent antibiotic activity against gram-positive bacteria by inhibiting dephosphorylation of lipid carriers.1, 3, 4 BA can directly disintegrate bacterial cytoplasmic membranes and has a profound effect on morphological structure and permeability of membranes and protoplasts, resulting in death of bacteria.5-7 In recent years, resistance to antibiotics has caused severe health problems. BA has the potential in combating resistant bacterial infection because resistance to BA is rarely found though it has been applied for several decades; the resistance cases to BA observed thus far are due to the degradation of the unique structural features protecting this polypeptide antibiotic by proteases.8 However, the narrow antibacterial spectrum and high nephrotoxicity of BA have limited its application in clinics. Hence, it is often used as only a last resort.9 Structure-activity relationship revealed modification of the polypeptide antibiotics by increasing hydrophobicity can improve membrane adsorption, insertion and the permeability of the antibiotics, which commonly translates into excellent antimicrobial activities.10 Many polymeric matrix materials, such as aliphatic acids,11,

12

palmitoyl groups,13 and cholesterol,14 have been

intensively investigated in this context. The introduction of polymeric matrix materials to antibiotics could enhance their desired antibiotic activity and lower their unwanted hemolytic activity. Hydrophobic modification can also endow polypeptide antibiotics spontaneously forming micelles, i.e., “nanoantibiotics”. Nanoantibiotics also contribute to the efficient administration of antibiotics by improving their pharmacokinetics and target accumulation, thus lowering their side effects.15-17 In our previous study, we explored the possibility of modifying BA with poly(D,L-lactic-co-glycolic acid) (PLGA) to impart antimicrobial properties and low toxicity.18 The results indicated that these PLGA-modified BA assemblies (Nano-BAPLGA) possessed a broader antimicrobial spectrum, stronger antibacterial activities and more satisfactory biocompatibility than their unassembled counterpart, BA, and the distribution of antibacterial activity was skewed toward longer PLGA blocks. However, the application of Nano-BAPLGA with longer PLGA chains was severely compromised by limiting water solubility. Poly(ethylene glycol) (PEG) is a polyether with high solubility in water.19 PEG can covalently 3



attach to hydrophobic copolymers, such as PLA, PLGA and PCA, producing copolymers with high solubility and ability of self-assembling.20,

21

Foreign particles, such as nanomedicines in the

bloodstream can be identified by the mononuclear phagocyte system (MPS) and then removed.22 By adding a polymer corona, i.e., “stealth sheath”, the foreign nature of a nanomedicine can be masked, thus exerting its antibacterial function before finally removed by the MPS. The commonly used “stealth sheath” is PEG. The procedure of adding a PEG corona to a drug is called “PEGylation”. In this study, we tried to develop nano-self-assemblies of BA with strong antibacterial properties, low toxicity, long circulation time and ability to target inflammatory tissue for potential application in the clinic in the future. PEGylated PLGA copolymers were chosen as the modified block because of their high solubility, long circulation time and good biocompatibility; these copolymers have been widely used in recent studies.23, 24 The antibacterial activities of PEGylated Nano-BAs were determined against a broad range of bacteria. The potential membrane destruction by PEGylated Nano-BAs was investigated by fluorescence spectroscopy, UV-VIS spectroscopy, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The behavior of PEGylated Nano-BAs with LPS was further analyzed to determine the function of LPS in bacterial resistance and to elucidate the underlying mechanism of PEGylated Nano-BA against gram-negative bacteria. In vivo imaging was carried out to verify the efficacy of the Nano-BAs in targeting inflammatory tissues. Eventually, a preliminary evaluation of the effectiveness of PEGylated Nano-BA treatment of infection burden in a thigh infection mouse model was conducted.

2. MATERIALS AND METHODS 2.1 Materials 2.1.1 Reagents PEG-PLGA-PEG triblock copolymers were purchased from Xi’an ruixi Biological Technology CO., Ltd. (Xi’an, China). N-phenyl-1-naphthylamine (NPN), 3,3’-dipropylthiadicarbocynine iodide (diSC3-5), o-nitrophenyl-β-D-galactopyranoside (ONPG),

rhodamine B isothiocyanate (RITC),

phosphatidylethanolamine (PE), cardiolipin (CL) and phosphatidylglycerol (PG) were purchased from Sigma-Aldrich (Shanghai, China). Bacitracin A was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). A LIVE/DEAD® BacLightTM Bacterial Viability Kit (L7012) was purchased from Thermo Fisher Scientific Inc. (Shanghai, China). Limulus amoebocyte lysate (LAL) 4


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kit was purchased from Xiamen BioEndo Technology, Co., Ltd. (Xiamen, China). 2.1.2 Bacteria and cells All bacteria used in this study and the human tubular epithelial cell line (HK-2) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Human red blood cells (hRBCs) were obtained from healthy blood donors. All blood donors provided informed consent for the use of their blood in this study. 2.1.3 Animals The experimental protocols used for the animal studies were approved by the Institutional Animal Care and Use Committee of Shenyang Agricultural University (Shenyang, China). Male Kunming (KM) mice weighing 20 ± 2 g were selected for the in vivo antibacterial activity experiments and the in vivo biodistribution study. Male Sprague-Dawley (SD) rats weighing 200 ± 20 g were selected for the in vivo pharmacokinetic studies. 2.1.4 Tested Formulations BA solution: bacitracin A solution; Nano-BAPLGA: nano-bacitracin A based mainly on BA-PLGA(5K)-BA;

Nano-BA5K:

nano-bacitracin

A

based

mainly

on

BA-PEG-PLGA(5K)-PEG-BA;

Nano-BA8K:

nano-bacitracin

A

based

mainly

on


Nano-BA10K:

nano-bacitracin

A

based

mainly

on

A

based

mainly

on


Nano-BA12K:

nano-bacitracin

BA-PEG-PLGA(12K)-PEG-BA. 2.2 Synthesis and Characterization of Copolymers All of the copolymers used in this work were constructed in our laboratory. The details of the synthesis and characterization of BA-PEG-PLGA-PEG-BA with different molecular weights of PLGA block are provided in the Supporting Information. 2.3 Preparation and Characterization of PEGylated Nano-BAs PEGylated Nano-BAs were prepared using a thin-film hydration method described in our previous study.18 The zeta potential, particle size and distributions of the PEGylated Nano-BAs were determined with a Zetasizer Naso ZS (Malvern, UK). The morphologies of the PEGylated Nano-BAs were observed after being stained with phosphotungstic acid using transmission electron microscopy (TEM, Hitachi HT-7700, Japan) at an acceleration voltage of 80 kV 2.4 In Vitro Antibacterial Activity Assays 5



The minimal inhibitory concentrations (MICs) of the Nano-BAs (Nano-BAPLGA, Nano-BA5K, Nano-BA8K, Nano-BA10K and Nano-BA12K) were determined using a modified microdilution method.25,

26

Briefly, a series of Nano-BA solution was obtained from the stock solution of

Nano-BAs (256 µM) by serial dilution. A bacterial suspension (100 µL, 106 CFU/mL) was added to 96-well microtiter plates, and then, 100 µL of Nano-BAs was added to each well. After incubation for 18 h at 37 °C, the inhibitory effect of Nano-BAs was evaluated by monitoring the absorbance at 600 nm on a multifunctional microplate reader (Tecan, Austria) 2.5 Confocal Laser Scanning Microscope Observations The viability of E. coli ATCC 25922 and S. aureus ATCC 29213 cells was measured using a LIVE/DEAD® BacLightTM Bacterial Viability Kit (L7012) after the Nano-BA treatment. Briefly, the E. coli (ca. 107 CFU/mL) and S. aureus (ca. 107 CFU/mL) cells were incubated with BA solution, polymyxin B, Nano-BAPLGA, Nano-BA5K, Nano-BA8K, Nano-BA10K or Nano-BA12K at 1× MIC for 4 h, respectively. Then, a dye mixture (SYTO 9 dye and propidium iodide (PI) in a ratio of 1:1) was added to the bacterial suspension (in a ratio of 3 µL: 1 mL). The bacterial suspension was incubated for another 15 min in the dark at room temperature and the microscopic images were taken using an Olympus FV1000-IX81 (Japan). The site targeted by Nano-BAs was also monitored using Confocal Laser Scanning Microscope (CLSM). Briefly, E. coli ATCC 25922 (ca. 107 CFU/mL) and S. aureus ATCC 29213 (ca. 107 CFU/mL) cells were incubated with RITC-loaded Nano-BA12K, RITC-loaded Nano-BA5K, RITC-loaded Nano-BAPLGA and RITC-labeled BA at 1/2× MIC for 0.5 h, 1 h, 2 h, 4 h and 8 h. Similarly, a smear was created, and microscopic images were taken by CLSM. The RITC-loaded Nano-BA preparation data are shown in the Supporting Information. 2.6 In Vitro Cytotoxicity The in vitro cytotoxicity of PEGylated Nano-BAs against HK-2 cells was assessed by a MTT assay. Briefly, HK-2 cells were seeded in 96-well plates. After incubation for 6 h, each well was added with fresh medium containing different concentrations of the tested formulations (Nano-BAPLGA, Nano-BA5K, Nano-BA8K, Nano-BA10K, Nano-BA12K and BA solution), and incubated at 37 °C for 24 h and 48 h, respectively. Then, for each well, MTT was added and the plates were incubated for another 4 h. A total of 150 µL of dimethyl sulphoxide was added to each well after the medium was completely removed. The absorbance at 570 nm was measured on a 6


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multifunctional microplate reader (Tecan, Austria). 2.7 PEGylated Nano-BAs Induced Membrane Disruption 2.7.1 SEM and TEM observations SEM and TEM observations were conducted with the same procedure in our previous study,18 while the details are provided in the Supporting Information. 2.7.2 Dye Leakage Assay Two types of liposomes with a PG/CL mass ratio of 3:1 and a PG/CL/PE mass ratio of 2:1:7 were prepared to mimic the S. aureus membrane and the E. coli membrane, respectively.27-29 The small unilamellar vesicles (SUVs) were prepared using a modified thin-film hydration method.30 Calcein release was detected after transferring 2 mL HEPES buffer solution (pH 7.4) and 4 mL of calcein-entrapped liposomes to a beaker under gentle stirring. Membrane permeation was detected by monitoring the fluorescence at λex (490 nm)/λem (520 nm) following the addition of Nano-BA12K, Nano-BA5K, Nano-BAPLGA, polymyxin B solution or BA solution. Triton X-100 (10% (v/v)) was added to ensure 100% dye release. The percentage of fluorescence intensity recovery, Ft, was calculated by eq. 1: = − / − × 100%

eq. 1

Where, It is the fluorescence intensity observed at equilibrium after addition of the tested formulations, I0 is the initial fluorescence intensity, and If is the total fluorescence intensity with the addition of Triton X-100. 2.7.3 Cytoplasmic Membrane Potential Measurement diSC3-5, a membrane potential-sensitive dye, was used for the determination of the cytoplasmic membrane electrical potential. Diluted E. coli ATCC 25922 and S. aureus ATCC 29213 cells in mid-log phase (with an OD600 of 0.2) were incubated with 4 µM diSC3-5. The fluorescence intensity at 670 nm was monitored after excitation at 622 nm. After diSC3-5 was fully absorbed by the bacteria, Nano-BA12K, Nano-BA5K, Nano-BAPLGA, BA solution or polymyxin B solution at a final concentration of 1× MIC was added to the bacterial samples. The fluorescence intensity was determined by an F-4500 fluorescence spectrophotometer (Hitachi, Japan) from 0 to 300 s. 2.7.4 Outer Membrane Permeability Assay The impact of Nano-BAs on the outer membrane permeability of bacteria was determined by the NPN uptake assay.25, 26, 31 E. coli ATCC 25922 and S. aureus ATCC 29213 cells (with 7


an


OD600 of 0.2) were added with 10 µM NPN and the background fluorescence was recorded (λex=350 nm, λem=420 nm) using an F-4500 fluorescence spectrophotometer (Hitachi, Japan). A total of 2 mL cell suspension was mixed with the tested formulations (Nano-BA12K, Nano-BA5K, Nano-BAPLGA and BA solution) in different concentrations, and the fluorescence intensity was recorded as a function of time. 2.7.5 Inner Membrane Permeability Assay Spheroplasts of E. coli were prepared by an osmotic shock procedure as previously reported.32 Using ONPG (1.5 mM) as the substrate, the release rate of β-galactosidase from spheroplasts of E. coli, intact E. coli and S. aureus cells (with an OD600 of 0.1) was measured to determine the inner membrane (cytoplasmic membrane) permeability of Nano-BAs .25 Subsequently, 2 mL cells were mixed with 1× MICs of Nano-BA12K, Nano-BA5K, Nano-BAPLGA, BA solution and polymyxin B solution, respectively, and incubated at 37 °C. The absorbance at OD420 were recorded every 2 min for a period of 30 min, and the permeability of the inner membrane was evaluated. 2.8 Interaction with LPS 2.8.1 LPS Neutralization LPS neutralization was evaluated using a quantitative chromogenic limulus amoebocyte lysate (LAL) kit. Stock solutions of Nano-BAs (Nano-BAPLGA, Nano-BA5K and Nano-BA12K) were prepared in pyrogen-free water. Nano-BAs at concentrations of 0, 1, 2, 4, 8, 16, 32, 64 and 128 µM were incubated with LPS (1 endotoxin unit) at 37 °C for 30 min. Then, an equal volume of LAL reagent was mixed with 50 µL of the mixture. After incubation for another 10 min, 100 µL of chromogenic substrate was added. The absorbance at A545 was measured at different concentrations of Nano-Bas, and the inhibition of LPS by Nano-BAs was evaluated. 2.8.2 LPS Binding Assay The binding affinities of Nano-BAs with LPS were detected using Zeta potential measurement. The zeta potential of LPS aggregates was measured in the presence of Nano-BAs at different concentrations (from 0 to 128 µM) using a Zetasizer Naso ZS (Malvern, UK). A blank measurement was set up in the absence of Nano-BAs. The concentration of LPS was set at 0.1 mM. 2.8.3 Disassociation of LPS DLS measurements were selected to determine the dissociation of LPS causing by Nano-BA12K. The hydrodynamic diameters of LPS after 1 h of incubation with or without 2 µM 8


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Nano-BA12K was measured on an NICOMPTM 380 ZLS (Santa Barbara, USA). The scattering data were collected at 90°. 2.9 In Vivo Pharmacokinetic Analysis Twenty-four Sprague-Dawley (SD) rats were randomly divided into four groups. Group I was received with BA solution as the control, Group II received Nano-BAPLGA, Group III received Nano-BA5K and Group IV received Nano-BA12K at a dose equivalent to 30 mg/kg via tail vein. Blood samples (0.5 mL) were drawn from orbit at predetermined time intervals: 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 36, 48 and 72 h after injection. Blood samples were centrifuged at 6000 rpm for 5 min. The clear supernatant was removed for further analysis. A liquid-liquid extraction method was used to extract drug from the plasma. The content of BA in blood samples was quantitatively analyzed by the HPLC/MS/MS method. The software DAS (version 2.1.1) was used to analyze the pharmacokinetic parameters. 2.10 In Vivo Biodistribution Study KM mice were inoculated by injecting 100 µL of 109 colony-forming units (CFU)/mL mixture of E. coli ATCC 25922 and S. aureus ATCC 2921333 into the thigh muscle of each hind leg to establish a mouse thigh infection model. The in vivo distribution of the Nano-BAs was assessed using the Kodak In Vivo Imaging System FX PRO (Carestream Health, Inc., USA). The near-infrared fluorescence dye DiR was selected for near-infrared (NIR) fluorescence imaging. Two days after infection, 0.2 mL of DiR-loaded Nano-BAPLGA (Nano-BAPLGA/DiR), DiR-loaded Nano-BA5K (Nano-BA5K/DiR) and Nano-BA12K (Nano-BA12K/DiR) was intravenously injected into the infected KM mice via tail vein. The time-dependent biodistribution in mice was imaged at 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h post-injection. The mice were sacrificed 48 h post-injection, and the main organs and thighs were harvested and rinsed with physiological saline. The fluorescence images of the organs were then acquired. The details of Nano-BAPLGA/DiR, Nano-BA5K/DiR and Nano-BA12K/DiR preparation are provided in the Supporting Information. The content of BA in the inflammatory site was further quantitatively measured by the HPLC/MS/MS method. Two days after infection, the mice were injected with the BA solution, Nano-BAPLGA, Nnao-BA5K or Nano-BA12K with an equivalent BA dose (30 mg/kg) via tail vein. At predetermined time points (4, 8, 12, 24, 36 and 48 h), the mice were sacrificed, infected thighs were harvested and weighed, and the BA content was quantitated by HPLC/MS/MS analysis. 9



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2.11 In Vivo Anti-infective Activity The mouse thigh infection model was established as described above. One hour after inoculation, the mice were intravenously injected with BA solution, polymyxin B solution, Nano-BAPLGA, Nano-BA5K or Nano-BA12K, respectively, at a dose of 30 mg/kg twice a day for 3 days. At 7 days after inoculation, the thighs were collected aseptically and weighed (0.5-0.7 g), before homogenized in 10 mL of sterile saline. The homogenate was diluted and incubated on agar plates overnight at 37 °C. The bacterial titer was expressed as l g CFU/g of thigh weight. The survival rate of the mice was monitored for 7 days post-infection. In addition, at the end of the experiment, blood samples were obtained for analysis of biochemical parameters by a 7100 Automatic Biochemical Analyzer (Hitachi, Japan). 2.12 Statistical Analysis Statistical analysis was performed using one-way variance (ANOVA) for more than three groups and Student’s t-test between two groups. Statistical significance was defined as *P < 0.05, and **P < 0.01.

3. Results 3.1 Characterization of the Nano-BAs The physical characterization of PEGylated Nano-BAs (Nano-BA5K, Nano-BA8K, Nano-BA10K and Nano-BA12K) is summarized in Table 1. The PEGylated Nano-BAs were approximately 100 nm in diameter, with excellent polydispersity indexes (less than 0.1). The TEM images show that the PEGylated Nano-BAs were spherical and homogenous, and the particle size was consistent with that obtained by DLS (Fig. 1). All of the Nano-BAs exhibited low zeta potential as evidenced by the slightly negative surface charge at pH 7.4. Table 1 The physicochemical characterization of Nano-BAs (n=3) Formulations

Particle size (nm)

ξ potential (mv)

PDI

Nano-BA12K Nano-BA10K Nano-BA8K Nano-BA5K

116.4±8.7 105.4±9.1 93.5±11.2 88.5±9.8

-4.21±0.09 -3.02±0.11 -2.89±0.14 -2.77±0.07

0.091±0.003 0.087±0.009 0.093±0.006 0.098±0.005

10


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Fig. 1 Morphology of Nano-BA12K (A), Nano-BA10K (B), Nano-BA8K (C) and Nano-BA5K (D) obtained by TEM after negative staining with 2 % sodium physphotungatate solution. Bar: 200 nm.

3.2 In Vitro Antibacterial Activities of the Nano-BAs The in vitro antibacterial activities of the PEG-PLGA-PEG copolymers and Nano-BAs (Nano-BAPLGA, Nano-BA5K, Nano-BA8K, Nano-BA10K and Nano-BA12K) against a series of gram-negative and gram-positive bacteria were determined. All of the copolymers showed no visible antibacterial activities (MICs>128 µM, see Supporting Information, Table S3). BA solution was only effective against gram-positive bacteria, and this result correlated well with those of previous studies.34-38 All of the tested PEGylated Nano-BAs, except Nano-BA5K, demonstrated strong antibacterial activities compared with Nano-BAPLGA, and Nano-BAs with longer PLGA chains were preferred. It is worth noting that Nano-BA12K even exhibited efficiency against gram-negative bacteria comparable to that of polymyxin B, a conventional gram-negative bactericide. However, when the length of the PLGA segment was further increased (Mn: 15 K), the antibacterial activities tended to level off, and the solubility sharply decreased (See Supporting Information, Table S3). Similar MIC values between Nano-BAPLGA and Nano-BA5K implied that PEGylation did not affect the antibacterial activities of Nano-BAs.

11



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Table 2 Antibacterial activities of the tested formulations (MIC) MICa (µM) Gram-positive

Formulations

Gram-negative

S. aureus

S. pyogenes

A. pyogenes

E. coli

P. aeruginosa

S. typhimurium

ATCC29213

ATCC19615

ATCC19411

ATCC25922

ATCC27853

ATCC13311

BA solution

2

4

4

>128

>128

>128

Nano-BAPLGA

1

2

2

4

8

8

Nano-BA5K

1

2

2

4

8

8

Nano-BA8K

0.5

1

1

2

4

8

Nano-BA10K

0.5

1

1

2

4

4

Nano-BA12K

0.5

0.5

1

1

2

2

Polymyxin B

>128

>128

>128

1

1

2

a

Minimal inhibitory concentrations (MICs) were determined as the lowest concentration of the tested Nano-BAs that inhibited bacterial growth.

The change in the viability of E. coli and S. aureus cells after incubation with PEGylated Nano-BAs was assessed using CLSM. The bacterial cells were stained with PI (red) and SYTO 9 (green) to visualize the dead and viable cell populations, respectively. All cells were stained by SYTO 9. However, only the cells with altered membrane permeability could be stained by PI. Thus, bacteria with intact cell membranes appeared to be green, whereas membrane-compromised bacteria appeared to be red. As illustrated in Fig. 2, compared to BA, all of the Nano-BAs displayed stronger red fluorescence and weaker green fluorescence for both E. coli and S. aureus cells, indicating lower viability. However, the fluorescence intensities of the Nano-BAs varied. The strongest red fluorescence was observed with Nano-BA12K, indicating the strongest bactericidal potency among the tested formulations. In addition, there was no obvious difference in fluorescent intensity between Nano-BA5K and Nano-BAPLGA.

12


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Fig. 2 The confocal microscope images of E. coli cells (A) and S. aureus cells (B) stained by LIVE/DEAD after incubation with Nano-BA12K, Nano-BA10K, Nano-BA8K, Nano-BA5K, Nano-BAPLGA, BA solution and Polymyxin B at 37 ºC. 3.3 In Vitro Cytotoxicity Assay

BA is not intended for systemic administration because it is nephrotoxic. To elucidate whether 13



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PLGA-induced self-assembly could reduce the cytotoxicity of BA, the in vitro cytotoxicity of the Nano-BAs (Nano-BAPLGA, Nano-BA5K, Nano-BA8K, Nano-BA10K and Nano-BA12K) against human HK-2 tubular epithelial cells (HK-2 cells) was measured by MTT assay (Table 3). All Nano-BAs exhibited higher IC50 values against HK-2 cells after 24 h incubation, indicating they had lower cytotoxicity. However, after incubation for 48 h, the IC50 values of the Nano-BAs significantly decreased, implying that cytotoxicity increased with the prolonged incubation times. Furthermore, PEGylated Nano-BAs displayed slightly lower cytostatic activity on HK-2 cells compared with non-PEGylated Nano-BAs during the whole incubation time. Table 3 IC50 values of BA solution and Nano-BAs against HK-2 cells (n=3) Formulations BA Solution Nano-BAPLGA Nano-BA5K Nano-BA8K Nano-BA10K Nano-BA12K

IC50 (µM) 24 h

48 h

4.36±0.47 27.24±1.22 28.32±1.43 29.55±0.98 30.21±1.37 30.54±0.86

4.18±0.29 4.59±0.98 6.14±1.12 6.29±1.01 6.20±0.89 6.17±0.91

3.4 Cellular Uptake The cellular uptake of Nano-BAs in S. aureus and E. coli cells was evaluated using CLSM. As shown in Fig. 3, compared with BA solution, all of the tested Nano-BAs exhibited stronger red fluorescence signals inside E. coli and S. aureus cells, indicating that the formation of self-assembled Nano-BAs could efficiently increase the uptake of BA by bacterial cells. BA solution exhibited no red fluorescence intensity in E. coli cells and weak red fluorescence intensity in S. aureus cells. Nano-BA12K resulted in the strongest fluorescence signal among the tested formulations. The fluorescence intensity increased from 0.5 h to 1 h and reached the maximum at 2 h. The relatively strong fluorescence lasted for more than 8 h. Compared with Nano-BA12K, even though Nano-BAPLGA and Nano-BA5K also revealed a broad distribution of red fluorescence in the cytosol, the fluorescence intense became much weaker after 4 h of incubation and almost disappeared after 8 h incubation. Moreover, Nano-BAPLGA and Nano-BA5K exhibited similar red fluorescence intensity during the observation time.

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Fig. 3 The confocal microscope images of E. coli cells (A) and S. aureus cells (B) incubated with RITC-loaded Nano-BA12K, RITC-loaded Nano-BA5K, RITC-loaded Nano-BAPLGA and RITC-labeled BA for 0.5 h, 1 h, 2 h, 4 h and 8 h at 37 ºC.

3.5 Assessment of the Effect of Nano-BAs on the Cell Membrane 3.5.1 SEM and TEM Observations SEM was first employed to observe the morphological structure of bacterial cells treated with 15



Nano-BAs. Compared with the negative control (Fig. 4A and 4G), the Nano-BAs induced a reduction of the cell membranes integrity of both E. coli and S. aureus. After the treatment with Nano-BAPLGA and Nano-BA5K, the surface of E. coli and S. aureus cells became rough but still retained cytoplasmic membrane integrity. Under the same treatment with Nano-BA12K, the E. coli cell membrane was disrupted, and protoplasts were exposed, leading to the promotion of aggregation (Fig. 4E). The S. aureus cells were also efficiently disrupted, while numerous pores were formed on the cell surfaces after incubation with Nano-BA12K (Fig. 4K). BA also induced a membrane-damaging effect on S. aureus cells (Fig. 4H), but failed to alter the morphology of E. coli cells (Fig. 4B).

Fig. 4 SEM micrographs of E.coli ATCC 25922 treated with negative control (A), BA Solution (B), Nano-BAPLGA (C), Nano-BA5K (D), Nano-BA12K (E) and Polymyxin B (F) for 1 h. SEM micrographs of S. aureus ATCC 29213 treated with Negative control (G), BA Solution (H), Nano-BAPLGA (I), Nano-BA5K (J), Nano-BA12K (K) and Polymyxin B (L) for 1 h. 16


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TEM was selected to further study the effects of Nano-BAs on the cell surface of bacterial cells. Without the Nano-BA treatment, both E. coli and S. aureus cells had a normal smooth surface. In contrast, the cells treated with 1× MIC of Nano-BAPLGA and Nano-BA5K demonstrated partial cell surface disruption with slight leakage of the cellular cytoplasmic content. The addition of Nano-BA12K to the bacteria caused significant morphological changes and complete cell surface disruption (Fig. 5E and Fig. 5K). The detachment of the outer membrane from the inner membrane of cells could be observed, with fragments of the cells remaining.

Fig. 5 TEM micrographs of E. coli ATCC 25922 treated with negative control (A), BA Solution (B), Nano-BAPLGA (C), Nano-BA5K (D), Nano-BA12K (E) and Polymyxin B (F) for 1 h. TEM micrographs of S. aureus ATCC 29213 treated with negative control (G), BA Solution (H), Nano-BAPLGA (I), Nano-BA5K (J), Nano-BA12K (K) and Polymyxin B (L) for 1 h. 17



3.5.2 Lipid Membrane Permeability The above electron microscopy observations proposed a hypothesis that the Nano-BAs might kill bacteria through disruption of the cell membrane/wall structure. To test this hypothesis, we constructed two liposomes (PG/CL/PE and PG/CL), resembling E. coli and S. aureus membranes, respectively.39 Membrane disruption was evaluated by measuring the release of calcein. As shown in Fig. 6A, out of these Nano-BAs, Nano-BA12K could rapidly induce calcein leakage (92%) from the PE/CL/PG liposomes in a time-dependent manner, which was similar to that induced by polymyxin B (94%), implying that the membranes of gram-negative bacteria were susceptible to Nano-BA12K. Nano-BA5K and Nano-BAPLGA could induce only 72% and 70% leakage from PE/CL/PG SUVs, respectively. BA solution was less effective than the Nano-BAs in terms of causing calcein leakage from the PG/CL/PE liposomes (48%). Similar results were obtained for PG/CL SUVs (Fig. 7B). The release of calcein induced by Nano-BA12K, Nano-BA5K, Nano-BAPLGA and BA solution was 94%, 73%, 74% and 64%, respectively. Complete leakage of calcein could be achieved by addition of Triton X-100.

Fig. 6 Nano-BA-induced calcein release as a function of time. Nano-BAs were added to PG/CL/PE SUVs (A) and PG/CL SUVs (B) encapsulated with calcein. Three independent trails were performed and the mean value was used for the graphs.

3.5.3 Membrane Depolarization The effect of Nano-BAs on membrane depolarization was evaluated by monitoring the fluorescence intensity change of the membrane potential-dependent probe diSC3-5. An increase in the fluorescence intensity indicates a reduction in the membrane potential. As shown in Fig. 7, the fluorescence signal of disC3-5 was not observed until the addition of Nano-BAs, which induced a rapid increase in the fluorescence intensity in both E. coli and S. aureus cells. However, the 18


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fluorescent intensities of the tested Nano-BAs varied. The membrane depolarization level of Nano-BA12K was significantly higher than those of Nano-BAPLGA and Nano-BA5K. In contrast, BA solution showed a moderate membrane depolarization effect in S. aureus cells and a very limited membrane depolarization effect in E. coli cells.

Fig. 7 Cytoplasmic membrane potential variation of E.coli (A) and S. aureus (B) treated with Nano-BA12K at 1× MICs was assessed by the release of disC3-5. The fluorescence intensity was monitored at λex=622 nm and λem=670 nm as a function of time. Three independent trails were performed and the mean value was used for the graphs.

3.5.4 Outer Membrane Permeability The permeability of Nano-BAs across the bacterial outer membrane was examined using an NPN uptake assay. The NPN fluorescent dye can enter cells with a disrupted outer membrane, as characterized by a marked increase in fluorescence. As shown in Fig. 8, Nano-BA12K caused a dramatic increase of the fluorescence intensity of NPN in E. coli and S. aureus cells in a concentration-dependent manner, indicating the LPS- or LTA-outer membrane/wall was at least partially disrupted. With the addition of 2 µM Nano-BA12K, the permeability of the outer membrane was more than 50% and increased over 90% when 4 µM Nano-BA12K was added. Comparatively, Nano-BAPLGA and Nano-BA5K induced a reduction in NPN uptake in E. coli and S. aureus cells at the same concentration. On the contrary, the BA solution was unable to disrupt the outer membrane of E. coli, which completely prevented NPN from entering the bacterial cells.

19



Fig. 8 The outer membrane permeability of the Nano-BAs. The uptake of NPN into E.coli (A) and S. aureus (B) cells in the presence of Nano-BAs at different concentrations was determined using the fluorescence dye assay. The NPN uptake was monitored at λex=350 nm and λem=420 nm. Three independent trails were performed and the mean value was used for the graphs.

3.5.5 Inner Membrane Permeability The ability of Nano-BAs to penetrate intact inner membranes of E. coli, spheroplasts of E. coli and S. aureus cells was further evaluated. As illustrated in Fig. 9, Nano-BA12K induced a rapid increase in absorption by intact E. coli, spheroplasts of E. coli and intact S. aureus cells at 1× MIC, similar to that of the positive control. Nano-BA5K and Nano-BAPLGA retained similar inner membrane permeability and slightly compromised permeability compared to Nano-BA12K. In contrast, BA solution showed a different capability in inducing the inner membrane permeability against intact E. coli and spheroplasts of E. coli. The inner membrane permeability of BA solution was considerably compromised against intact E. coli within 100 min, but significantly enhanced against spheroplasts of E. coli. This observation indicated that the outer membrane of E. coli cells might be a formidable barrier for BA molecules to overcome, while Nano-BAs could cross this barrier.

Fig. 9 Effect of the Nano-BAs on the permeability of the inner membrane of E. coli cells (A), spheroplasts of E. coli cells (B) and S. aureus cells (C). Three independent trails were performed and the mean value was used for the graphs. 20


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3.6 Interaction of Nano-BAs with LPS 3.6.1 LPS Neutralization The potent effect of Nano-BAs on LPS neutralization was determined by a LAL assay. Nano-BA12K at a concentration of 16 µM could inhibit the LAL enzymes mediated by LPS and nearly 80% of the endotoxin. Nano-BAPLGA and Nano-BA5K, played a similar role at a higher concentration of 32 µM. However, the BA solution was unable to neutralize LPS. 3.6.2 Binding of Nano-BAs to LPS The binding ability of Nano-BAs with LPS was detected by measuring the change of zeta potential caused by LPS. As shown in Fig. 10B, a negative zeta potential of -38.6 mv was observed for the suspension of E. coli cells. The addition of Nano-BA12K could effectively neutralize the negative charge on the bacterial surface. When the concentration of Nano-BA12K was above 4 µM, the zeta potential was dramatic increased, suggesting the binding with LPS was effective. When the concentration of Nano-BA12K continued to increase to more than 32 µM, a negative charge (-4.33 mv) close to the zeta potential of Nano-BA12K (-4.21 mv, Table 1) was detected, indicating Nano-BA12K was inserted into LPS. Nano-BAPLGA and Nano-BA5K were also effective at neutralizing LPS but at a higher concentration. 3.6.3 Disassociation of LPS The structural perturbation upon the addition of Nano-BAs was measured using fluorescent FITC-conjugated LPS (Fig. 10C). FITC fluorescence was self-quenched, and the disaggregation of LPS was evaluated by the changes of the fluorescence intensity.40 As illustrated in Fig. 10C, the fluorescence intensity was enhanced by the addition of Nano-BAs, which was due to the probable dissociation of LPS. Furthermore, Nano-BAs displayed a concentration-dependent manner. The fluorescence intensity improved with the increasing of Nano-BA12K until the concentration reached 16 µM. When the concentration was higher than 16 µM, the FITC fluorescence intensity leveled off, suggesting the complete disassociation of the LPS aggregates. Compared with Nano-BA12K, the disassociation ability of Nano-BAPLGA and Nano-BA5K was slightly compromised, presenting as complete disassociation of LPS at a higher concentration of 32 µM. The disaggregation of LPS caused by Nano-BA12K was further confirmed by DLS studies. Aggregates of LPS alone had a diameter of approximate 7000 nm (Fig. 10D). Adding Nano-BA12K could cause a dramatic shift of the size of the LPS aggregate from 7000 nm to 2000 nm (Fig. 10E). 21



The peak appearing at approximately 150 nm corresponded to Nano-BA12K.

Fig. 10 LPS neutralization by Nano-BAs. The figure shows the inhibition of LPS at 1 EU/mL by Nano-BAs at ten different concentrations (A). The effect of Nano-BAs on the charge of the surface of E. coli ATCC 25922 cells (B). Disaggregation of LPS by Nano-BAs. The changes of fluorescence intensity of FITC-conjugated LPS in the presence of Nano-BAs (C). Size distribution of LPS (D) and size distribution of LPS in the presence of Nano-BA12K (E). Data are plotted as mean ± SD obtained from six independent observations.

3.7 In Vivo Pharmacokinetics and Biodistribution Fig. S5 shows the concentration of BA in rats after intravenous administration of the tested formulations, including BA solution, Nano-BAPLGA, Nano-BA5K and Nano-BA12K. The corresponding pharmacokinetic parameters are listed in Table 4. Compared with BA solution, all of the Nano-BAs could significantly increase the circulation time in the body. The elimination half-time (t1/2β) of Nano-BAPLGA, Nano-BA5K and Nano-BA12K was markedly increased from 5.618 h to 11.367 h, 15.428 h and 21.298 h, respectively. The area under the plasma concentration-time curve (AUC) for Nano-BAPLGA, Nano-BA5K and Nano-BA12K was 2171.135, 3792.14 and 5244.86 ng·h/L, respectively, which was 3.54-, 6.19- and 8.56-fold higher than that of BA solution (612.942 22


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ng·h/L), respectively. Meanwhile, the MRT values for Nano-BA12K, Nano-BA5K and Nano-BAPLGA were 2.1-fold, 1.8-fold and 1.5-fold higher than that of BA solution, suggesting a marked delay in blood clearance. Moreover, compared with Nano-BAPLGA, the AUC of Nano-BA5K and Nano-BA12K was markedly increased. Table 4 Comparative pharmacokinetic parameters of the BA formulations (n=6) Parameters t1/2 (β) (h) AUC0→t (ng/L/h) AUC0→∞(ng/L/h) MRT0→∞(h) CL (L/h/Kg) V1 (L/Kg)

Formulations BA solution

Nano-BAPLGA

Nano-BA5K

Nano-BA12K

5.618 553.392 612.942 10.12 0.038 0.207

11.367 2155.595 2171.135 15.506 0.009 0.126

15.428 3688.21 3792.14 17.954 0.006 0.113

21.298 5028.827 5244.86 21.949 0.003 0.097

The time-dependent biodistribution and targeting efficiency of Nano-BAs containing near-infrared dye DiR were evaluated in a mouse thigh infection model using an optical image system. As shown in Fig. 11A, the Nano-BA12K/DiR showed effective inflammatory tissue targeting and relative longer residence at inflammatory tissue. The fluorescence reached the maximum at 12 h post-injection, and the fluorescence lasted for more than 48 h. Nano-BA5K/DiR was also effective in targeted delivery to inflammatory tissue but to a lesser degree compared with Nano-BA12K. In addition, stronger fluorescence signal in liver was observed at all time points when compared with that of Nano-BA12K. Nano-BAPLGA/DiR showed lower inflammatory targeting and a shorter circulating time than PEGylated Nano-BAs, which might be due to uptake by the RES system. The precise targeting ability of Nano-BAs was further verified by ex vivo imaging of the thigh tissues after 48 h post-injection. As shown in Fig. 11B, Nano-BA12K/DiR showed the strongest fluorescence intensity in inflammatory tissue among the tested formulations. Excised organs were also harvested and imaged (Fig. 11B). Intense fluorescence signaling was observed in the liver and spleen, corresponding to the main clearance route of nanoparticles.41 Nano-BA12K resulted in the weakest fluorescence signal in kidney among the tested groups, indicating lower nephrotoxicity. The amount of BA in inflammatory sites was quantified through a validated HPLC/MS/MS method (Fig. 11C). For the free BA, only a low concentration of BA was detected in the thigh tissue, indicating that free BA was unable to target the inflammatory tissue. In contrast, BA could effectively accumulate into the inflammatory tissue when delivered via Nano-BAs. Compared with non-PEGylated Nano-BAPLGA, PEGylated Nano-BA12K and Nano-BA5K were more efficient at 23



targeted delivery of BA into the inflammatory tissue, which was consistent with the in vivo imaging results. Furthermore, the content of BA in inflammatory tissue delivered by Nano-BA5K was lower than that delivered by Nano-BA12K.

Fig. 11 The in vivo non-invasive images of thigh burden infection KM mice after i.v. injection of Nano-BAPLGA, Nano-BA5K and Nano-BA12K (A). The ex vivo optical images of thigh tissue and organs of bacteria infected mice sacrificed at 48 h after i.v. injection of Nano-BAPLGA, Nano-BA5K and Nano-BA12K (B). Quantitative analysis of BA accumulation in inflammatory tissue in thigh burden infection KM mice at different times after intravenous administration of BA solution, Nano-BAPLGA, Nano-BA5K and Nano-BA12K at a dose of 30 mg/kg, respectively (C).

3.8 In Vivo Anti-infective Activity of Nano-BAs With the promising in vitro results presented above, we proceeded to evaluate the efficacy of the Nano-BAs in treating KM mice with infected thighs in vivo. At 7 days post-infection, thigh tissues were removed to determine the quantitative counts of bacteria (CFU). As shown in Fig. 12A and B, all of the Nano-BAs resulted in significant decreases in bacterial counts. Nano-BA12K resulted in the eradication of nearly 4.91 log10 viable CFU of E. coli (P

PEGylated Self-Assembled Nano-Bacitracin A - ACS Publications

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