Integrated Endotoxin Adsorption and Antibacterial Properties of

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Biological and Medical Applications of Materials and Interfaces

Integrated Endotoxin Adsorption and Antibacterial Properties of Cationic Polyurethane Foams for Wound Healing Yingying Ding, Zhe Sun, Rongwei Shi, Hengqing Cui, Yangyang Liu, Hailei Mao, Bin Wang, Duming Zhu, and Feng Yan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19746 • Publication Date (Web): 26 Dec 2018 Downloaded from http://pubs.acs.org on December 27, 2018

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ACS Applied Materials & Interfaces

Integrated Endotoxin Adsorption and Antibacterial Properties of Cationic Polyurethane Foams for Wound Healing Yingying Ding a,†, Zhe Sun b,†, Rongwei Shi b, Hengqing Cui c, Yangyang Liu a, Hailei Mao a,*, Bin Wang c,*, Duming Zhu a,*, Feng Yan b a

Department of Anesthesiology and Critical Care Medicine, Zhongshan Hospital,

Fudan University, Shanghai 200032, China b

Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application,

Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China c

Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s

Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, China †Authors with equal contributions.

E-mail: [email protected]; [email protected]; [email protected]

1

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Abstract: Gram-negative bacteria, containing toxic proinflammatory and pyrogenic substances (endotoxin or lipopolysaccharide (LPS)), can lead to infection and associated serious diseases, such as sepsis and septic shock. Development of antimicrobial materials with intrinsically endotoxin adsorption activity can prevent the release of bacterial toxic components while killing bacteria. Herein, a series of imidazolium-type polyurethane (PU) foams with antimicrobial properties were synthesized. The content effects of cationic moieties on the antimicrobial activities against gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), and gram-positive Staphylococcus aureus (S. aureus), as well as the endotoxin adsorption property were investigated. The obtained PU foams show slightly higher efficiency against two gram-negative strains than for gram-positive one, and high absorbability of LPS. A wound healing test using P. aeruginosa and its isolated LPS treated mice as the models further demonstrated that imidazolium-type PU foams combine both antibacterial and endotoxin-adsorption properties, and may have potential application as an antimicrobial wound dressing in a clinical setting.

Keywords: Endotoxin adsorption; Antibacterial; Imidazolium cation; Polyurethane foam; Mouse model.

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Introduction With the widespread use of antibiotics, the issue of bacterial resistance has become a global threat and challenge.1,2 Among 12 "superbugs" listed by the World Health Organization (WHO) in 2017, three gram-negative bacteria, including Acinetobacter Bauman, Pseudomonas aeruginosa and Enterobacteriaceae, were classified as “critical”, the highest risk category. Gram-negative bacteria are not only susceptible to drug resistance, but their cell walls also contain a common characteristic pathogenic component, lipopolysaccharide (LPS), also known as endotoxin, a highly toxic inflammatory and pyrogenic substance.3,4 LPS can activate the intracellular NF-κB-mediated inflammatory pathway through the cell membrane Toll-like receptors, after being released from the destructed bacterial cell wall.5-9 As a result, gram-negative bacteria and their constituent LPS can lead to wound infection, pneumonia, sepsis and other severe infection associated diseases,3 suggesting that it is indispensable to consider the treatment of bacterial toxic ingredients while killing the bacteria. LPS is exposed on the outer leaflet of the outer membrane of gram-negative microbes, and is made up of three layers from inside to outside:10,11 i) Lipid A is the major source of LPS toxicity, two outer phosphorylated GlcN (glucosamine) units make the molecule negatively charged, and the inner multiple fatty acids anchor the whole LPS into the bacterial outer membrane; ii) The core polysaccharide layer: the inside has two negatively charged KDO (ketodeoxyoctulosonate) molecules by covalent binding with one GlcN of lipid A; the outside contains eight sugar units, which is also with negative charges; iii) O antigen is composed of different monosaccharides, for example, most of P. aeruginosa has two glyforms: the neutral CPA (common polysaccharide antigen, also called A-band) and the negatively 3



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charged OSA (O-specific antigen, B-band).12 In a word, the negative charge of LPS molecules can not only contribute greatly to the structural integrity of bacteria, and also provide the potential property for the removal of their own.13 To our knowledge, the medical endotoxin adsorbents are mainly aimed at LPS that has already been in the bloodstream.14-16 A variety of polymeric materials, such as membrane and fiber, have been developed to remove endotoxin by means of hemofiltration or dialysis.17,18 The design of these materials is mainly based on the principle of affinity chromatography, so that a plenty of ligands specific binding to LPS are screened out for the removal of endotoxin, including polymyxin B,17,19-21 histamine,22 histidine,23 deoxycholic acid, amines,24 amino acids,25-27 polymerized cations28-30 and so on. Compared with polymyxin B and other small ligands, cationic polymers have the advantages of strong specific adsorption to LPS molecules, such as weak nonspecific adsorption with proteins, and good biocompatibility.31-34 The main mechanism for endotoxin removal involves the electrostatic force between positive cations of these polymers and negative charged LPS,35 which is also fit for the explanation of antimicrobial property, indicating that it is possible to develop some kind of antimicrobial materials with intrinsically endotoxin adsorption activity. However, the existing cationic polymers, such as polyethyleneimine,36,37 poly (L-lysine),38 and poly (L-histidine), are limited for application due to the instability and (or) relatively high cost.39 In this study, a series of cationic polyurethane (PU) foams were synthesized. The effects of cation content of PU foams on the antimicrobial activities against gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), and gram-positive Staphylococcus aureus (S. aureus), and the endotoxin adsorption property using the limulus amoebocyte lysate (LAL) quantitative assay 4


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were investigated. The obtained PU foams show slightly higher antibacterial efficiency against two gram-negative strains than for gram-positive one, and high absorbability of LPS. On the basis of in vitro antimicrobial and biocompatibility, one foam containing 9 wt% of imidazolium ionic diol was evaluated for the repairing of a skin wound predisposed with P. aeruginosa PAO1 and its isolated LPS in a mouse model. The results demonstrated that imidazolium-type PU foams combine both antibacterial and endotoxin-adsorption properties, and may have potential application as an antimicrobial wound dressing in a clinical setting.

Materials and methods Materials. 2-Bromoethanol, 1-(2-hydroxyethyl)imidazole, ethyl acetate, acetonitrile, methanol, polypropylene glycol (PPG) (molecular weight: ~3000 g/mol), and toluene diisocyanate (TDI) were purchased from Shanghai Chemical Reagents Co. (Shanghai, China). Strains of S. aureus (ATCC 6538) and E. coli (8099) were provided by Dr. Shengwen Shao (Huzhou University School of Medicine, China). Strain of P. aeruginosa PAO1 (ATCC 15692) was purchased from Biobworg Co. (Beijing, China). DNase, RNase and protease were purchased from Solarbio Science & Technology Co. Ltd. (Beijing, China). The limulus amoebocyte lysate (LAL) assay was provided by Zhanjiang A & C Biological Ltd. (Zhanjiang, China). ELISA assays were got from Boster Biological Technology Co. Ltd. (Wuhan, China). Human dermal fibroblasts were provided by Shanghai Ninth People’s Hospital of China. All reagents were analytic grade and used as received without further purification. The water used was deionized throughout the experiments.

Synthesis of imidazolium ionic diol. 5



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1,3-Bis(2-hydroxyethyl) imidazolium bromide was synthesized by stirring a mixture

containing

bromoethanol

(11.41

g,

0.105

mol)

and

1-(2-hydroxyethyl)imidazole (11.20 g, 0.100 mol) in 70 mL acetonitrile solution in inert atmosphere at 50 °C for 24 h. After the evaporation of solvent, the resultant viscous oil was washed with diethyl ether three times, and then dried in dynamic vacuum at 80 oC for 24 h before use (yield: 83%). 1H NMR (400 MHz, D2O, δ ppm): 7.56 (m, 2H), 4.33 (m, 4H), 3.92 (m, 4H) (see Figure S1).

Preparation of cationic polyurethane foams. A mixture containing imidazolium ionic diol (2 mmol for Foam 1, 4 mmol for Foam 2, and 6 mmol for Foam 3, respectively), PPG (1 mmol), water (5 mmol), poly[dimethylsiloxane-co-[3-(2-(2-hydroxyethoxy)ethoxy)propyl] methylsiloxane] (as surfactant, 0.03g) and dibutiltindilaurate (as catalyst, 0.002 g) was stirred at room temperature for 2 min (see Table 1). Upon the addition of isocyanate, the mixture was vigorously stirred for another 10-20 seconds, and then stored at room temperature to allow the formation of foam (see Scheme 1 and Table 1).

Table 1. Formulations for the Preparation of Three Foams with Different Content of Cationic Imidazolium Diol. Ionic Diol (mmoL)

PPG (mmoL)

TDI Water (mmoL) (mmoL)

Catalyst (g)

Surfactant (g)

Foam 1

2

1

9

5

0.002

0.03

Foam 2

4

1

11

5

0.002

0.03

Foam 3

6

1

13

5

0.002

0.03

6


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Scheme 1. Syntheses of Polyurethane Foams with Cationic Imidazolium Diol (The insert is the sectional photograph of prepared cationic foam).

Characterization. 1H

NMR spectra were recorded on a Varian 400 MHz spectrometer at 400 MHz

using D2O as the deuterated solvent of the imidazolium bromide. Soild-state 1H NMR spectra of synthesized foams were tested by AVANCEIII/WB-400. Fourier transform infrared (FT-IR) spectra were recorded on a Specode 75 model spectrometer in the range of 4000-400 cm-1. The mass spectra were detected via the electrospray ionization mass spectrometry (ESI-MS, Aligent 1200/6220). Scanning electron microscopy (SEM) images were taken with a Philips Model XL 30 FEG microscope with an accelerating voltage of 10 kV. The energy-dispersive X-ray spectroscopy (EDX) measurements were performed with a spectrometer attached to a Hitachi Model S-4700 field-emission scanning electron microscopy (FE-SEM).

Colony assay for the antimicrobial activities. 7



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Three microbes, S. aureus, E. coli and P. aeruginosa, were used to detect the antimicrobial ability of cationic polyurethane foams. Firstly, the bacteria were inoculated in Luria−Bertani (LB) medium, and cultured overnight at 37 °C, with 150 rpm of shaking, until the exponential growth phase reached. Then 400 μL of bacterial suspensions (diluted to about 1 × 106 /mL) was added into the sterilized foam samples (1×1×0.2 cm3), and incubated at 37 °C with a relative humidity higher than 90 %. To measure the antimicrobial activity of each foam sample, 10 μL microbial suspension was spread onto LB agar plate to count the colony formation units (CFUs) after 4 h of incubation. The colony assay was carried out at least three times independently. Common polyurethane foams without any positive ions were used as controls.

Extraction of LPS from P. aeruginosa. The P. aeruginosa strain PAO1 was cultured in 1L of LB medium to the value of OD600 up to ~ 1.0. The ultrasonication method prescribed by Darveau and Hancock was used for LPS preparation in this study.40 Briefly, the bacterial cells were destroyed by sonication, and the nucleic acids of PAO1 decomposed by a mixture of DNase and RNase, and the bacterial proteins digested by protease. Finally, LPS was obtained by ultracentrifugation. After separated by 12% SDS-PAGE gel, the LPS constituent components extracted from PAO1 were then identified by silver staining.41 To quantify the prepared LPS of PAO1, the limulus amoebocyte lysate (LAL) assay was applied. Finally, the LPS samples were stored in PBS at -20 °C for further use.

Endotoxin adsorption assay. LPS from PAO1 was diluted to the endotoxin content (C0) of 2 EU/mL and 15 8


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EU/mL, respectively. After sterilized at 121 °C for 4 h to inactivate the residual LPS, the foam samples (1×1×0.2 cm3, about 0.08 g for each) were put into 6 mL of LPS solutions with two concentrations. Time course of endotoxin content (Ct) in solution was quantified using LAL assay at 5 min, 30 min, and 60 min, respectively. According to the equation: (C0-Ct)/C0×100%, the endotoxin adsorption rates were calculated after the foam samples were put into the LPS solutions for 1 h. The independent experiments were performed in triplicate.

Computer simulation of endotoxin adsorption mechanism. The simulations were carried out with the GROMACS package version 5.1.2, using CHARMM36 force field for the outer membrane of Gram-negative bacteria P. aeruginosa. For modeling cationic polyurethane foams, the parameters were generated from the Antechamber and ACPYPE tools, and the RESP charges were derived from ab initio calculations performed at B3LYP/6-31+g(d) level of theory using Gaussian03 software. According to the molecular structure and the content of imidazolium ionic diol shown in Scheme 1 and Table 1, two simulated models (Foam 1 and Foam 3, with m=1 and n=4) were constructed by saturating terminals with OH groups or adding cationic imidazolium idol groups to keep total charge to be +1e and +3e, respectively. Finally, two interaction models of P. aeruginosa outer membrane with Foam 1 and Foam 3 were set up: the former consists of 30 Foam 1 structures with 30 positive charges, the latter contains 20 Foam 3 units with 60 positive charges, and both foams were randomly placed 0.8 nm near the surface of the pre-equilibrated P. aeruginosa outer membrane, which was composed of 5 LPS molecules and 143 DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) lipids (see Figure S11). The rough LPS molecule was modelled to contain 10 negative charges using CHARMM-GUI 9



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Membrane Builder. All the calculations were referenced to the previous studies.42,43 The detailed simulations were performed as follows: the particle-mesh Ewald method was used to calculate the long-range electrostatic interactions, the Lennard-Jones potential and forces were truncated at 1.2 nm, and the bond lengths were constrained by PLINCS with 2 fs time step. Each system was first equilibrated using an isochoric-isothermal (NVT) ensemble for 6 ns with the Berendsen weak coupling algorithm and a coupling constant of 0.1 ps, and then equilibrated under an isobaric-isothermal (NPT) ensemble for 10 ns with the coupling constants for temperature and pressure of 0.2 ps and 2.0 ps, respectively. After that, the unconstrained production simulations were performed for 600 ns using a Nose-Hoover thermostat and a Parrinello-Rahman barostat. During all simulations, the temperature was maintained at 310 K.

Cytotoxicity evaluation. The

cytotoxicity

of

foam

samples

was

determined

by

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. In brief, human dermal fibroblasts (~ 3×104) in 1 mL Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum were cultured in a 24-well plate for 72 h, and then the sterilized foam samples (1.0×1.0×0.2 cm3) were immersed in the fibroblast cell solutions. After the incubation at 37 °C for 72 h, 100 µL of MTT (5 mg/mL, in PBS) was added into each well for another 4 h. Then, the culture media was discarded and 750 µL of DMSO was added in each well, which was incubated at room temperature for 30 min to dissolve the formazan crystal products. The cell viability was evaluated by the absorbance of each well at 490 nm on the Eon microplate spectrophotometer (Bio-Tek instruments, Inc), and the relative growth 10


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rate (RGR) was calculated by the equation of OD sample/OD control ×100% with common polyurethane foam without any positive ions as a control. The measurements were repeated three times independently.

Hemolysis assay. The fresh human blood samples from healthy volunteers were centrifuged at 1500 rpm for 15 min to harvest red blood cells (RBC), and then the RBC samples were carefully washed with PBS until the supernatant was transparent and then diluted to 2 vol% in PBS for further usage. After the sterilized foam (1.5×1.5×0.2 cm3) was dipped into 4 mL of diluted RBC suspension for each tube and incubated at 37 °C for 3 h, the treated blood samples were centrifuged at 1500 rpm for 15 min, and then aliquots of 100 μL supernatant were transferred to a 96-well plate for measure of the absorbance at 576 nm on the Eon microplate spectrophotometer (Bio-Tek instruments, Inc). The diluted RBC suspensions in 2 % Triton and in PBS were applied as the positive and negative controls, respectively. The hemolysis rate was calculated by the formula of (OD sample–OD negative control) / (OD positive control–OD negative control) ×100%. The independent experiments were performed in triplicate.

Water absorption and air permeability of foams. Each foam (2×2×0.2 cm3) was dried at 55 °C overnight and weighed accurately (M1) before tests. After put into 10mL of water for 4 h, the foam was taken out and weighed again (M2). Then, the water absorption of each sample was calculated by the equation of (M2-M1)/M1×100%. To measure the air permeability of foam samples, a wide mouth bottle was poured into 50 mL of water and weighed accurately (G), and then the bottle was covered with 11



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a foam at the mouth, and placed at 37 °C with the humidity of 75% for 24 hours (weighted as G1). The air permeability rate (%) =(G1- G) / (G control - G) ×100%, while the bottle without any foam was used as a control (G control).

In vivo wound healing study. The ethical committee of the Zhongshan Hospital approved all the experimental protocols in accordance with the national regulations on animal studies. Male BALB/c mice (6-8 weeks old) were housed in a temperature-controlled room with 12-hour periods of light-dark exposure. After the mouse was anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg), an open excision wound (1.5×1.5 cm2) without full thickness skin was created on the dorsal side. Fifty eight mice were randomly assigned to five groups. The solutions of P. aeruginosa PAO1 (109 CFUs/mL) or LPS extracted from equal amount of PAO1 (4×104 EU/mL) were respectively used as two positive controls for the wound healing assay of the foams, and PBS as a negative control (100 μL for each solution). The wounds of all controls were covered with sterile gauze directly. While the PAO1 or LPS infected wounds covered with Foam 3 (2.0×2.0×0.2 cm3) were classified as two test groups. Ten mice were used in PBS negative control group, and twelve mice in other four groups, including PAO1 and LPS positive controls, and their corresponding foam-treated test groups. On the 0th, 4th, 7th and 14th post-operative days, the appearance of the wounds was photographed after the dressings removed. The unhealed wound rate was calculated by the percentage of At/A0, where At and A0 represent the wound areas on the specified day and the day of operation, respectively. On day 14th after surgery, the entire wound with adjacent normal skin was excised and fixed in 4% 12


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paraformaldehyde, and observed with the hematoxylin and eosin (H&E) staining for the further histological analysis. The number of inflammatory cells in the wound area was counted on the H&E stained sections by the ImageJ 1.41 software. After 12-hour post operation, the overall status of mice was recorded by a digital camera. To further evaluate the systemic inflammation response and organ functions, blood samples were collected at 6 h, 12 h, 24 h, and 48 h after surgery, respectively. Four mice were included for each time point in every group. The levels of TNF-α (Tumor necrosis factor-α), IL-6 (Interleukin-6) and IL-1β (Interleukin-1β) were detected by ELISA. The indicators to liver and kidney functions, including AST (Aspartate transaminase), ALT (Alanine transaminase), Cr (Creatinine) and BUN (Blood urea nitrogen) were tested by the biochemical method. In addition, the body weight and survival rate of mice were recorded during the process.

Statistical analyses. Statistical analyses were performed by GraphPad Prism 6.0 (GraphPad, Inc., La Jolla, CA, United States). All numeric data were presented as mean ± SD. The unpaired Student’s t test was used to compare the difference; P < 0.05 was considered to be statistically significant.

Results and discussion Preparation and characterization of imidazolium-type polyurethane foams. Scheme 1 depicts the chemical structures of imidazolium diol and the synthetic route to the imidazolium-type cationic PU foams. The purity and chemical structures of ionic diol, 1,3-bis(2-hydroxyethyl) imidazolium bromide, were confirmed by 1H NMR and MS spectra, respectively (as shown in Figure S1-2). The PU foams were 13



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prepared by stirring a mixture containing imidazolium diols, PPG, TDI, tin catalyst, silicone-based surfactant, and water at room temperature.44-46 The obtained cream grew to foams at room temperature. The inserted sectional photograph of prepared cationic foam in Scheme 1 shows the digital photographs of obtained PU foams. The surfaces of sponge pores are smooth, and no obvious changes were observed for the variation of imidazolium ionic idol amount. Similar results were obtained by SEM images. As can be seen that all the pores are full and even (Figure S3). Furthermore, all the free-standing foams are soft enough to be cut into any desired size. The synthesized PU foams were characterized by means of FT-IR spectra, using Foam 3 as a representative sample (Figure S4). Two absorption peaks at around 1597 and 752 cm−1 arise from the vibrational mode of imidazolium cations. The absorption peak at around 3295 cm−1 attributed to the stretching vibration of N−H, while the peak at around 1098 cm−1 is the characteristic peak of C−O−C. The peaks at 2866-2968 cm−1 attribute to the stretching vibration of C−H. The absorption peaks at 1716 and 1642 cm−1 belong to the urethane and urea C=O. Figure S5 shows the solid-state 1H NMR spectra of imidazolium-based Foam 3. The peak attributes to the hydrogen of the alkyl chain linked to imidazolium cation was observed. The Energy dispersive X-ray (EDX) spectra (Figure S6) shows a higher N atom content of Foam 3, as compared with the PU foam without imidazolium cations. All these results confirmed the successful preparation of imidazolium-type PU foams.

Antibacterial activity of imidazolium-type polyurethane foams. The antibacterial properties of imidazolium-type PU foams was first investigated. Two gram-negative (E. coli and P. aeruginosa) and one gram-positive (S. aureus) 14


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bacteria were chosen as model microorganisms. The bacterial proliferation was tested by colony assay at various times. As it can be seen from Figure 1 and Figure S7 that all three foams display certain antibacterial activities after contacting with three microorganisms for 4 h. It is not a surprise that the antimicrobial efficiencies of the synthesized PU foams increase with the content of cationic groups. Among the PU foams synthesized, Foam 3 showed the highest antimicrobial efficiencies. The viable colonies of E. coli, P. aeruginosa and S. aureus almost completely disappeared after 4 h contacting with Foam 3 (see Figure 1A-C and Figure S7). The result was further supported by the dynamic variation of the antibacterial process (see Figure 1A’-C’). In addition, the antimicrobial efficiencies of these foams on gram-negative bacteria are higher than on gram-positive ones. These results indicate that the strong electrostatic interaction between imidazolium cations and negative charged cell wall are main antimicrobial force. The antibacterial mechanisms of the cationic compounds (polymers) usually based on the electrostatic interactions between the cationic moieties and negative charged of the bacterial cell membranes.28,47 On the other hand, the mechanism for endotoxin removal also involves the electrostatic force between positive cations of these polymers and negative charged LPS35, indicating that it is possible to develop antimicrobial materials with intrinsically endotoxin adsorption activity.31-34

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Figure 1. Bacterial viabilities of E. coli (A), P. aeruginosa (B), and S. aureus (C), after contacting with cationic polyurethane foams for 4 h. Common polyurethane foams without positive ions were used as controls (left column). Line charts show the time course of surviving E.coli (A′), P. aeruginosa (B′), and S. aureus (C′) upon contacting with the foams (right column).

Endotoxin adsorption efficacy of imidazolium-type polyurethane foams. As it is well known that endotoxin or LPS is the characteristic component of gram-negative bacteria. In this study, we selected P. aeruginosa PAO1 for the 16


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extraction of its lipopolysaccharide, not only due to the effective antibacterial properties of the foams to PAO1, but also for the reason that PAO1 was originated from the infected wound of a clinical patient, thus more fit for the subsequent in-vivo study.48 The lipopolysaccharide of PAO1 was extracted using the improved hot phenol method, and detected by silver staining following the separation in a SDS-PAGE gel. The main LPS constituent components, O-antigen and core-lipid A regions in Figure 2B also displayed the purity of our extracted LPS products.

Figure 2. (A) Digital photographs of three cationic polyurethane foams and the control one. (B) Electrophoretic profile of lipopolysaccharide (LPS) extracted from P. aeruginosa PAO1 by silver staining. The main LPS constituent components, O-antigen and core-lipid A regions were indicated. (C) Time course of LPS levels in solutions quantified using limulus amebocyte lysate (LAL) assay and corresponding LPS adsorption rates calculated after the foam samples were put into the extracted LPS solutions of 2EU/mL and 15EU/mL, respectively.

To evaluate the endotoxin adsorption of the foams to be tested (see Figure 2A), the solutions of two given concentrations, 2 EU/mL and 15 EU/mL of LPS from PAO1, 17



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were prepared, and then the time course of LPS levels in solutions was quantified using limulus amoebocyte lysate (LAL) assay after soaking the sponge in a solution for 1 h. The rapid decrease of LPS levels in solution in Figure 2C indicating that the cationic foams may be able to adsorb or destroy the LPS molecules. LPS is quite stable and extremely difficult to be destroyed, therefore, the removal of LPS remains to be a great challenge in the field of biomedicine. Here, the adsorption of LPS by PU foams could be the major reason for the reduced LPS levels in solution. According to the decreased velocity of LPS, esp. at 5 min, Foam 3 shows the highest efficiency for the adsorption of LPS in solution. The result consistent with the antibacterial activity (see Figure 2C and Figure 1B). The result means that electrostatic force between imidazolium cations and negative charged LPS molecules plays the same key role in endotoxin adsorption and microbe killing. The endotoxin adsorption rates of three foams at 60 min are all more than 95% in both concentrations of 2 EU/mL and 15 EU/mL of LPS, Foam 3 attains to 99.38% adsorption even to high concentration of endotoxin (see Figure 2C). All the results above display that Foam 3 is a good candidate for antibacterial materials combined with the rapid and effective adsorption of endotoxin released from the decomposed bacteria, so as to prevent endotoxin from entering the body.

Computer simulation of endotoxin adsorption mechanism of imidazolium-type polyurethane foams. To interpret the endotoxin adsorption mechanism of the cationic foams, a model of P. aeruginosa bacterial outer membrane was constructed by five anionic LPS molecules on the outer leaflet and the inner lipid bilayer, consisting of 143 zwitterionic DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) molecules. It can be 18


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easily observed that the foam molecules (Foam 1 and Foam 3) are gradually close to the surface of the bacterial outer membrane, and LPSs are pulled and even inserted into the space of foam molecules with the increasing of the simulation times (see Figure 3A-C and 3E-G). Moreover, the analyses of energy between the different components display that the effects mainly originate from the interactions between foams and LPS, and the electrostatic interactions are stronger than the hydrophobic ones (see Table 2, Figure 3D and 3D, and Figure S12). Obviously, the remarkable electrostatic interaction of -229.24, -394.28 kcal/mol (see Table 2) induced by the negatively charged LPS and positively charged imidazolium cations is the main drive force for the endotoxin adsorption. Meanwhile, the hydrophobic interaction in the endotoxin adsorption efficacy cannot be ignored because it occupies ~30% of the total interaction energies between Foam-LPS (see Table 2), consistent with the previous report.49 By comparison to Foam 1, Foam 3 can interact with the bacterial outer membrane more rapidly and stronger. As shown in Figure 3D and 3H, when the molecular model of bacterial outer membrane was placed near Foam 3, it was immediately disturbed by the fluctuation of the interaction energy with LPS; whereas for Foam 1, the membrane disturbance appeared after 50 ns. Moreover, Foam 3 forms higher total interaction (-563.25 kcal/mol) with endotoxin than that (-322.02 kcal/mol) of Foam 1 (see Table 2). These theoretical results agree well with the above experimental data that Foam 3 has the best endotoxin adsorption and antimicrobial activities (see Figure 1, Figure S7 and Figure 2C).

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Figure 3. Computer simulation of endotoxin adsorption mechanism displaying that the snapshots for the interaction process of P. aeruginosa outer membrane (LPS molecules and lipid bilayer) with Foam 1 (A-C) and Foam 3 (E-F) at different simulation times (0 ns, 240 ns and 600 ns), and the interaction energy changes between Foam 1 (D) or Foam 3 (H) and LPS during the whole simulation. Foam 1 and Foam 3 are labeled with light grey and dark grey respectively, LPS molecules are shown in magenta sticks, and the lipid bilayer is in green lines. Ele represents the electrostatic interaction and Hyd means the hydrophobic interaction.

Table 2. Mean Interaction Energies for LPS and DOPC Components of P. aeruginosa Outer Membrane with Foam 1 or Foam 3 during the Whole 600 ns Simulation. Interaction components

Electrostatic interaction Hydrophobic interaction Total interaction (Ele, kcal/mol) (Hyd, kcal/mol) (kcal/mol)

Foam 1-LPS

-229.24

-92.78

-322.02

Foam 3-LPS

-394.28

-168.97

-563.25

Foam 1-DOPC

-20.99

-17.01

-38.00

Foam 3-DOPC

-28.09

-22.78

-50.87

Biocompatibility and physical properties of imidazolium-type polyurethane foams. The biocompatibility of materials is essential for medical applications. Herein, the in vitro cytotoxicity of all foams was evaluated by MTT and hemolysis assays, 20


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respectively. As it can be seen in Figure 4A that all the imidazolium cationic foams are qualified for RGR values (higher than 75%). The hemolysis rates of foams are lower than 5%. Foam 3 shows the highest biocompatibility, indicating that incorporation of imidazolium cation could reduce the toxicity of foams to both human dermal fibroblasts and blood red cells as we expected.

Figure 4. (A) Relative growth rate (RGR) and hemolysis rate of cationic polyurethane foams. The toxicities to both human dermal fibroblasts and blood red cells were detected by MTT and hemolysis assays, respectively. Common polyurethane foams without cationic ions were used as controls. (B) Water absorbability and air permeability of cationic polyurethane foams. Common polyurethane foams without cationic ions were used as controls.

It is also important to take into consideration of the physical properties of materials as dressings. In this study, both water absorbability and air permeability of imidazolium cationic foams are measured. As shown in Figure 4B that Foam 3 has a little higher water content and air permeability rate than other foams, suggesting that Foam 3 can be applied for infected wounds as a new dressing, by combination of the antibacterial and endotoxin adsorption performance.

Wound healing test using P. aeruginosa PAO1 and LPS infected mice.

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Based on the in vitro antimicrobial activity, endotoxin adsorption, and cytotoxicity evaluations studied above, Foam 3 was chosen for the further wound healing test using a mouse model. Skin wound in experimental mice is a simple and commonly used model for in vivo evaluation of biomaterials. Gram-negative P. aeruginosa is one predominant pathogen to skin wound. With the spread of drug-resistant bacteria, P. aeruginosa has become one of the major global public health concerns for its highest critical rating by WHO in 2017. Therefore, P. aeruginosa PAO1 and LPS extracted from equal amount of PAO1 were used for wound healing assay of the sponge. The wounds with the same areas on the adult male mice treated with PAO1 or LPS and PBS, and then covered with sterile gauze directly were set as positive and negative controls, respectively. Whereas the PAO1 or LPS predisposed wounds covered with Foam 3 were classified as two test groups. As shown in Figure S8 for the wound healing evaluation of mouse model during 14 post-operative days, on the Day 0 and Day 1, no visible difference in wound appearance was observed for all the mice. From the Day 4 on, only the positive controls with PAO1 present a wound festering and become more obvious on the Day 7, whereas the other positive controls treated with LPS also experience a delayed healing on the Day 14, almost the same as the PAO1 infected wounds, suggesting that LPS act as a causative factor in the pathogenic process of Gram-negative bacteria. After covered with Foam 3 on wounds, both PAO1 and LPS treated wound surfaces present almost the same healing process and remain neat and tidy as the PBS negative controls. Correspondingly, on Day 4, both Foam 3 applied groups exhibit a reduced unhealed wound area than that of the PAO1 or LPS positive group covered with sterile gauze directly. On Day 14, such a downward trend is growing, about 30 % lower than that of the PAO1 and LPS positive controls. In particular, the group of 22


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LPS treated with Foam3 almost has no any unhealed wound area, and repair the wound as good as that of the PBS negative group (see Figure S8A-B). In addition, the average wound healing days for Foam 3 treated mice are significantly reduced as compared with those of PAO1 or LPS inoculated mice, that is, 17 ± 1.22 (PAO1 + Foam 3) and 14.75 ± 1.48 (LPS + Foam 3), respectively, the former is a little longer and the latter is nearly the same days for PBS negative controls (12.5 ± 1.12 days). However, it took about 22.25 ± 1.23 days and 21.75 ± 1.92 days for PAO1 and LPS positive controls to heal the skin wounds, respectively. (see Figure S8C). The delayed wound healing in both PAO1 and LPS positive groups should be due to bacteria and their toxic components, obviously, the role of LPS is impressive. Therefore, the fast wound healing observed in two foam-treated groups can be attributed to the bactericidal and LPS-adsorption effects of Foam 3. These results indicate that imidazolium ionic idol-based foams synthesized in this work are efficacious for PAO1 and LPS predisposed wounds, consistent with their corresponding in-vitro performance (see Figure 1 and Figure 2). It is not surprise that the efficacy of Foam 3 for LPS is better than that for PAO1, since LPS is only one major toxic component of PAO1, other bacterial components and products play more different roles in the pathogenicity of P. aeruginosa, including outer membrane proteins, cilia, Pseudomonas exotoxin A (PEA), phospholipase C and Pseudomonas quinolone signal (PQS) etc.50-53 Histological examination in the full-thickness wounds was also performed to record the microscopic appearances of local skins for each group on the 14th day. Although the representative wounds treated with and without Foam 3 all appear well developed new epidermis and dermis as the PBS negative ones (see Figure 5A-E), the inflammatory cells infiltrated in the subcutaneous areas in two foam-treated wounds 23



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are far less than positive controls, though a little more than PBS negative controls (see Figure 5F), which is consistent with their individual performance for the wound healing days (see Figure S8C). The effective healing in histology indicates both the imidazolium ionic idol-based foams can be used for infectious wound applications by killing microbes and removing endotoxin.

Figure 5. Representative histological analysis of skin wound by H&E staining on 14th day post operation from PAO1 (A) and LPS (C) positive controls and their individual Foam 3 treated mice (corresponding to B and D, respectively), and PBS negative control (E). The infiltrated inflammatory cells in wounds of five groups were counted (F). Data were presented as mean ± SD, n=4 for each group, ** represents P < 0.01, *** represents P < 0.001.

24


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Figure 6. Overall assessment of model mice. (A-E) Representative images for animal systemic status at 12 hours post operation from PAO1 (A) and LPS (C) positive controls and their individual Foam 3 treated mice (corresponding to B and D, respectively), and PBS negative control (E). The inserted pictures are enlarged to display the inflammatory reaction of eyes in five groups. The body weight (F) and survival rate (G) of mice in five groups for wound healing test. Data were presented as mean ±SD, n=12 in each groups. To further assess the biomedical performance and biosafety of imidazolium-type foams for wound healing, the animal systemic status along with the indicators for systemic inflammation reaction and organ functions in mice were observed. Figure 6A-E shows the representative images for animal systemic status at 12 hours post operation in five groups. The PAO1 infected mouse is the most sluggish, the hair is 25



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loose and grey, and the eyes are darkish and sunken and fail to open. As for LPS treated mouse, the systemic symptoms seem a little slight, however, the eyes are full of secretions and open incompletely. After applying the foams, the systemic status in two treatment groups is relieved, it is remarkable that the LPS mouse treated with Foam3 almost appears to the same as the PBS negative one. It indicates that the inflammation response induced by LPS on the local wound can be systematic with LPS rushing into the bloodstream, and appear rapidly and early before 12h. This is in line with the clinical principle that the infected wound must be treated as soon as possible. The wound dressing with the fast and strong efficacies of antimicrobial and endotoxin-adsorption will be more useful and worthy of recommendation for the removal of endotoxin with the bacteria killed and decomposed. Figure S9 shows that the serum levels of inflammatory factors, including TNF-α, IL-1β and IL-6 in two foam-treated groups, are significantly down-regulated during the first two-day post operation as compared to their individual positive controls with PAO1 and LPS, and are a little closer to those of negative controls with PBS. Moreover, the peak time of IL-1β is about at 12h post operation, while TNF-α and IL-6 elevate as early as 6h possible. More concretely, the PAO1 infected mice produce more IL-1β in sera than LPS ones, on the contrary, the LPS treated mice have the higher IL-6 and TNF-α levels, it indicates that the systemic inflammation response to LPS in body is quite rapid by the promotion of IL-6 and TNF-α, consistent with the systematic symptoms mentioned above (see Figure 6A-E). The similar changes can also be observed in the indicators for liver (AST and ALT) and kidney (BUN and Cr) functions, including the significant decrease in two foam-treated groups compared to their positive controls with PAO1 and LPS, and a little more close to those of negative controls with PBS (see Figure S10). However, 26


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the peak time of these indicators for organ function is a little later at about 12h post operation as that for IL-1β (see Figure S10), suggesting that the organ dysfunction be more likely due to the consequence of inflammatory factor stimulation. Therefore, it is crucial for the design of antimicrobial dressing to adsorb endotoxin released from the killed bacteria to reduce the bacteria and their toxic constituents into the bloodstream, thus, in turn, the systemic inflammatory response provoked by LPS and subsequently the initiation of organ dysfunction can be avoided. In similar, the body weight change and survival rate in PAO1 and LPS groups treated with foams are close to the PBS negative controls for wound healing test (see Figure 6F-G), also providing the additional evidence for the antibacterial and endotoxin-adsorption performance, and biosafety of the imidazolium ionic diol-based foams.

Conclusions In this study, the imidazolium-type cationic PU foams were successfully prepared and characterized with both antimicrobial and endotoxin adsorption properties. These foams show high antimicrobial and endotoxin adsorption performance. Furthermore, wound healing tests using both P. aeruginosa bacteria and their LPS treated mice as the models demonstrated the imidazolium-type PU foams can be developed as the new type antibacterial dressings with intrinsically endotoxin adsorption activity for clinic applications.

Supporting Information.  Characterization of imidazolium-type PU foams.  Antimicrobial characterization of imidazolium-type PU foams by in vitro and in 27



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vivo assays.  Computer simulation of endotoxin adsorption mechanism of imidazolium-type PU foams.

Acknowledgements This work was supported by the National Science Foundation for Distinguished Young Scholars (No. 21425417), the Natural Science Foundation of China (No. 21704071, U1862109, 81571930, 81772115), General Program Foundation of Shanghai Municipal Commission of Health and Family Planning (201740107), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Science and Technology Innovation Fund Project of Zhongshan Hospital, Fudan University (2017ZSCX03).

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Integrated Endotoxin Adsorption and Antibacterial Properties of

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