Fabrication of polylysine based antibacterial coating for catheters by facile electrostatic interaction

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Fabrication of polylysine based antibacterial coating for catheters by facile electrostatic interaction ⁎

⁎

Huan Yua,b, Lin Liua, Xue Lib, , Rongtao Zhoua, Shunjie Yana, Chunsheng Lib, Shifang Luana, , ⁎ Jinghua Yina, Hengchong Shia, a

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical Materials, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China

b

H I GH L IG H T S

method was used to fabricate antibacterial catheter based on ε-poly-ʟ-lysine complex coating. • AThisfacile coating exhibits excellent antibacterial activity and biocompatibility both in vitro and in vivo. • The coating with multi-performances has promising potential for biomedical devices. •

A R T I C LE I N FO

A B S T R A C T

Keywords: Indwelling catheter-related infections ε-Poly-ʟ-lysine (PL) Antibacterial coating Biocompatibility Electrostatic interaction

Despite the development of advanced antibacterial biomedical materials, bacterial infection is still a serious problem for indwelling catheter because it usually induces severe complications. Hence, medical indwelling catheter with the capabilities of antibacterial activity, stability, biocompatibility is urgently needed. In this work, a water-insoluble antibacterial coating based on ε-Poly-ʟ-lysine (PL) was prepared via a facile electrostatic interaction between cation PL and anion surfactant, 1,4-bis(2-ethylhexyl) sodium sulfosuccinate (AOT). The ease and efficacy of the PL-AOT complex preparation render it as antibacterial coating applicable to a variety of medical devices for reduction of bacterial infections. This coating was fabricated on the medical catheter with broad-spectrum antibacterial activity, long-term stability, biocompatibility. The contact-killing oriented strategy for the antibacterial action of this coating was confirmed by high-performance liquid chromatography (HPLC). Almost 100% S. aureus and E. coli as Gram-positive and Gram-negative bacteria model could be killed rapidly by this coating for thermoplastic polyurethane (TPU) film. The antibacterial properties of the coated catheters were also assessed under static and dynamic flow conditions. Regardless of the above conditions, the coated catheters displayed remarkable antibacterial activity compared to the uncoated catheters. In addition, this coating showed better antibacterial stability by mimicking the in vivo environment, that is, the antibacterial efficacy could still maintain even after 31 days immersing. Moreover, the coated catheter exhibited negligible cytotoxicity against L929 murine fibroblasts cells. For in vivo experiment, the coated catheter caused 90% less inflammation in mice and showed more remarkable antibacterial performance. Consequently, this ε-Poly-ʟ-lysine (PL) based coating have a great potential to serve as a safe and multifunctional antibacterial strategy for the medical indwelling devices.

1. Introduction Bacterial colonization of implantable medical devices could cause severe public health care problem owing to the associated risks of infection, high cost of treatment, and development of drug resistance. Nosocomial infections are considered as a global health challenge

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which is the sixth leading cause of death [1]. The indwelling catheterrelated infections are frequently associated with microorganisms, including the catheter-associated urinary tract infections (CAUTIs) and catheter-related bloodstream infections (CRBSIs), which represent severe medical problems [2,3]. An estimated 4% to 10% prevalence rate of nosocomial infections in western-industrialized countries caused by

Corresponding authors. E-mail addresses: [email protected] (X. Li), sfl[email protected] (S. Luan), [email protected] (H. Shi).

https://doi.org/10.1016/j.cej.2018.10.160 Received 26 May 2018; Received in revised form 10 September 2018; Accepted 20 October 2018 1385-8947/ © 2018 Elsevier B.V. All rights reserved.

Please cite this article as: Yu, H., Chemical Engineering Journal, https://doi.org/10.1016/j.cej.2018.10.160

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polymer chains separated by layers of surfactant [29,30]. Although several types of antibacterial PSCs have been synthesized, there have no work that apply PSCs based on ε-Poly-ʟ-lysine (PL) to the medical catheter surface for building an antibacterial coating surface. The PL consisting 25–35 L-lysine residues has been widely used in many countries such as Japan and U.S.A that is approved by the Food and Drug Administration (FDA) as the food preservatives in 2003 [31], in addition, the PL was certified that have highest antibacterial activity toward the foodborne bacteria such as E. coli, S. aureus by destroying the cell membrane [32,33]. More than anything else, compared to other bactericide, PL shows no side effects on the human [34,35]. The PL has also been approved as a food additive in China in 2014. However, the insolubility of PL in almost all common organic solvents limits its application, especially as coating precursors. The PL exists in polycationic form by the protonation of ε-amino groups under in acidic and neutral conditions, thereby, we expected that by introducing anionic charge with hydrophobicity into PL, the water insoluble and organo-soluble complex with an antibacterial activity could be developed. Herein, we report a contact-killing oriented bactericidal coating based on organo-soluble ε-Poly-ʟ-lysine (PL) and 1,4-bis (2-ethylhexyl) sodium sulfosuccinate (AOT) complex (PL-AOT), which could be noncovalently immobilized onto catheter surfaces without tedious preparation process. The ease and efficacy of the PL-AOT complex preparation render this it as antibacterial coating with broad-spectrum antibacterial activity, good biocompatiblity, long-term stability. The PL-AOT coated catheters exhibited better antibacterial properties under static and dynamic flow condition. Finally, in vivo experiments were also carried out to validate the tissue compatibility and antibacterial activity of the coating.

bacterial colonization of a broad range of biomedical surfaces, the rate reaching up to 30% specially in the intensive care units [4–6]. Moreover, the proportion is typically higher more than 15% in the developing world [7]. Therefore, prevention of indwelling catheter-related infections is one of the important challenges to the medical community. Bacterial attachment and the subsequent proliferation and colonization on the surfaces of biomaterials such as indwelling catheter usually result in the formation of a biofilm [8–11]. Bacterial adhesion is detrimental to the function of the indwelling catheter and limits its service life [12,13]. The biomaterial surfaces are rapidly fouled and coated with biological fluids such as plasma proteins, which is facilitate to bacterial attachment [14,15]. In order to prevent the biofilm formation, it is essential to build an effective antibacterial catheter surface [15,16]. Leong et al. firstly employed a methodic approach that combined a simple selective chemical immobilization platform developed on a silicone catheter with the choice of a potent antimicrobial peptide (AMP), to allow site specific immobilization of AMP at an effective surface concentration. The AMP coated catheter demonstrated strong anti-adhesive properties and good antibacterial against both Gram positive and negative bacteria [17]. Furthermore, Leong’s team developed a peptide-immobilized PD coating which applied to polydimethylsiloxane (PDMS) and commercially available Foley catheter recently. The coating providing the surfaces with potent antimicrobial and antibiofilm properties against relevant UTI-causing bacteria, with better stability and biocompatibility properties [18]. Kizhakkedathu et al. reported a polymer brush based implant coating that was nontoxic, antimicrobial and biofilm resistant. These coating consists of covalently grafted hydrophilic polymer chains conjugated with an optimized series of AMPs [19]. Based on novel polymer-based tethering strategy, this team also developed a very effective antimicrobial coating consisting of highly active AMPs attached to PU catheter surfaces that not only had non-fouling characteristics but also provided specific flexible binding sites for peptide conjugation [20]. Although remarkable progress has been made in the development of the antibacterial surfaces, limitations also existed [13]. Therefore, it is still a challenge for construction of antibacterial catheter with efficacy, long-term stability, cost-effectiveness, due to the special shape of the catheter and different application conditions. Surface coating, graft polymerization and bulk modification are usually adopted to achieve antibacterial catheter construction. Previous attempts to develop antibacterial catheter by surface coating have shown significant limitations, such as the instability of the coating, short-term antibacterial activity and efficacy against only a limited spectrum of bacterial species, all of which limit the use of such coatings for both the short- and long-term catheterization [21]. For graft polymerization methods, the several synthetic steps, using of harsh reagents, specific surface pretreatments and elevated temperatures, achieving the high surface packing densities and remaining long-term stability of biological material are enormous challenges [22]. For the bulk modification scheme, the coordination between the mechanical properties of the bulk and the antibacterial ability should be taken into consideration [23] and the complicated process are needed, especially in industrial production. Catheter surfaces coated with an effective antibacterial agent have been considered a promising approach to thwart the microbial infections as the method does not alter the bulk properties of materials [24]. Hence, it is necessary to construct an antibacterial catheter surface with better antibacterial property, good biocompatibility, long-term stability which is simply prepared. Polyelectrolyte-surfactant complexes (PSCs) have stimulated a great deal of interest in the last three decades, due to its importance in fundamental polymer physics, biological systems, nanotechnology, medicine, food science and industrial applications [25–27]. The stoichiometric PSCs are formed when equimolar amounts of charged polymer chain units and surfactant molecules are mixed in water [28]. These complexes are water-insoluble and in the solid state assemble spontaneously into lamellar structures consisting of alternating layers of

2. Experimental 2.1. Materials ε-Poly-ʟ-lysine (PL, Molecular weight: 3500–5000 Da) was purchased from Nanjing Bioshineking Biotech Co., Ltd. 1,4-bis(2-ethylhexyl) sodium sulfosuccinate (AOT, 96%) was purchased from Aladdin. All other chemicals (AR grade) were used as-received directly without further purification. The test films with thickness of 1 mm and medical grade catheter (OD: 2.5 mm, ID: 1 mm) used for the in vitro study were made of thermoplastic polyurethane (TPU, 1190A) from BASF Company, I.V. Catheter (TPU, 24G) used for in vivo test was made by ourselves. Gram-negative Escherichia coli (E. coli, ATCC 25922) and Gram-positive Staphylococcus aureus (S. aureus, ATCC 6538) were obtained from Nanjing Clinic Biological Technology Co. Ltd. L929 murine fibroblasts cell line was obtained from Shanghai ASTRI Cell Resource Center, Chinese Academy of Sciences. Cell Counting Kit-8 (CCK-8) was purchased from Boster Biological Technology Co. Ltd. H&E (Hematoxylin and Eosin) Staining Kit was purchased from Beijing Solarbio Technology Co., Ltd. 2.2. Synthesis and characterization of the PL-AOT complex The PL-AOT complex was prepared by dropwise addition of isovolumetric AOT solution (3% (w/v) AOT in 60% ethanol) into PL solution (1.5% (w/v) PL in 0.2 M of HCl) under stirring. The mixture was centrifuged at 10000 rpm for 15 min. The obtained white precipitate was washed three times with ultrapure water and dried in vacuum freeze dryer for 8 h (Supplementary Scheme S1). 1 H NMR spectroscopy was carried out using a Bruker AV 400 MHz spectrometer with d-dimethyl sulfoxide (d-DMSO) as a solvent. In brief, 5 mg PL-AOT complex was dissolved in 500 μL d-DMSO. 2.3. Preparation of PL-AOT coated samples The TPU films and catheters were washed with 75% ethanol for 2

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2.6. The antibacterial activity of intraluminal catheter under static and dynamic flow condition

15 min under ultrasonication, and then immersed in 1 mL of 2% (w/v) PL, AOT and PL-AOT complex ethanol solutions for 1 min, respectively. All samples were dried at ambient temperature and washed with ultrapure water of 37 °C for three times. For the convenience of description, the film and catheter samples were marked as TPU (unmodified), TPU/PL, TPU/AOT, TPU/PL-AOT and Cat (unmodified), Cat/PL, Cat/ AOT, Cat/PL-AOT, respectively.

Medical TPU catheters were sterilized (120 °C, 30 min) and dried overnight. The catheter was cut into fragments with length of 12 cm, and coated with the PL-AOT. In order to explore the antibacterial activity of the coated catheter under static condition, the S. aureus bacterial suspension (100 μL, 1 × 106 CFU mL−1) was injected into the intraductal catheter and incubated at 37 °C for 3 h. After incubation, 20 μL of the bacterial inoculum was extracted and plated for colony counts. The 4 mL fresh LB medium was added to the remaining inoculant as described above. The mixed bacterial suspension was incubated at 37 °C with shaking. Optical density measurement (OD540) with TECAN absorbance reader (TECAN GENIOS, Austria) were conducted at regular intervals to test the bacterial growth. Dynamic flow condition assays were performed on a microfluidic cultivation system, which mimics the actual environment of indwelling devices in vivo (Supplementary Scheme S2). The PL-AOT coated and uncoated catheters were filled up with S. aureus suspension (50 μL, 1 × 106 CFU mL−1) and incubated at 37 °C for 3 h to simulate bacteria adhesion. After the initial attachment phase, the catheter was connected to the infusion bag and incubated in the fresh LB medium at a constant flow rate of 0.1 mL/min at 37 °C. The medium was refreshed every 24 h. The catheters were collected at different time points. Adhered live bacteria inside catheters were quantitatively determined by colony counts. The number and morphology of the adherent bacteria were also evaluated using SEM.

2.4. Surface characterization of TPU/PL-AOT 2.4.1. Contact angle measurement TPU/PL-AOT samples were subjected to a sessile-drop method with a contact-angle goniometer drop-shape analysis (KRÜSS GMBH, Germany) at room temperature. A 2 μL droplet of water was dropped onto the dry film surface with a microsyringe and allowed to spread out across the surface for 1 min, and then the water contact angles (WCAs) of the samples were recorded. All samples are in triplicate for calculating the average value. 2.4.2. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy ATR-FTIR spectra were obtained from a FTIR spectrometer (BRUKER Vertex 70) with an ATR mode. A total of 32 scans were accumulated with a resolution of 4 cm−1 for each spectrum. 2.4.3. X-ray Photoelectron spectroscopy (XPS) The elemental composition was performed on X-ray Photoelectron Spectroscopy (XPS, VG Scientific ESCA MK II Thermo Avantage V 3.20 analyzer) with an Al Kα source (hν = 1486.6 eV). Binding energy range from 0 to 1200 eV was averaged at each point to identify potential elements on the sample surface.

2.7. The long-term antibacterial stability assay and the antibacterial mode investigation of PL-AOT coated catheter The 0.9% NaCl aqueous solution (stroke-physiological saline solution) is often applied in physiological experiments or clinical trials, because the osmotic pressure is equal to that of human plasma and tissue fluid. Therefore, in order to explore the coated catheters had good stability in the simulated human environment, the coated and uncoated catheters were immersed in 0.9% NaCl aqueous solution for different days. In short, the samples were immersed in 0.9% NaCl aqueous solution for 1, 3, 7, 14, 21 and 31 days and subjected to antibacterial assay via OD540 (Part 2.6). Briefly, the catheters collected at different days were injected with the S. aureus (1 × 106 CFU mL−1) and incubated at 37 °C for 3 h. The bacterial inoculum was immersed in fresh medium and incubated at 37 °C with shaking subsequently. After incubation for 18 h, the 200 μL bacterial suspension was transferred to 96-well plates and measured with OD540. The long-term antibacterial stability of the coated catheter in the urine (Dongguan Xinheng Technology Co., Ltd.) as the specific body fluids was also conducted as described above. To further make sure the antibacterial mode, ultra-pure water was injected into the coated catheter and leaching for 24 h. The extract solution as described above was subjected to detection via HPLC measurement.

2.5. The antibacterial activity of PL-AOT coated films An overnight S. aureus suspension was inoculated in 50 mL of LB medium, and cultured overnight at 37 °C with shaking. The culture solution was centrifuged (3000 rpm, 10 min), and the deposited bacteria were conducted to obtain a final concentration of 106 cells mL−1 via serial dilution. Agar plate colony counting assay: The antibacterial activity of the coating was quantitatively evaluated using viable cell count method, according to JIS Z 2801 standard. Briefly, TPU/PL-AOT and respective control films with the size of 1.5 × 1.5 cm were placed in a 6-well plate and 25 μL of S. aureus suspension were added at the center region of the plate. Subsequently, the S. aureus suspension was covered with PE film (1 × 1 cm). After cultivated at 37 °C for 24 h, the samples were immersed in 3 mL of PBS buffer and ultrasonicated for 2 min to release the adherent bacteria into PBS solution. Then the bacteria suspension was diluted and plated for colony counts at 37 °C for 24 h. The bactericidal activity value (R) was calculated using Eq. (1), A and B represent the average number of viable bacteria obtained after 24 h inoculation of the antibacterial sample and the control sample, respectively.

R= log

B A

2.8. Cytotoxicity and Hemolysis assay

(1)

The morphology of bacteria was also observed under field emitted scanning electron microscopy (SEM, XL 30 FESEM FEG, FEI Company, USA). The samples (1.0 × 1.3 cm) were placed in 24-well plates and covered with the S. aureus bacterial solution (106 cells mL−1, 1 mL). After cultivated at 37 °C for 24 h, the samples were removed to new 24well plates and washed with PBS buffer for three times to remove loosely adherent bacteria and fixed in paraformaldehyde (4 wt%, 1 mL) for 30 min. After fixation, the samples were washed three times with PBS and ultrapure water respectively. The morphologies of S. aureus were observed with SEM. All tests were carried out in triplicate. The surface antibacterial assay was also repeated for E. coli.

The cytotoxicity of TPU and TPU/PL-AOT samples were investigated by using the CCK-8 assay, according to ISO 10993-5 standards. L929 murine fibroblasts cell was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 vol% fetal bovine serum (FBS), and 1 vol% penicillin–streptomycin solution. The cells were detached from the culture flask by addition of 0.25% trypsin-EDTA solution, and resuspended in fresh medium for subsequent experiments. Cells in culture medium (100 μL) at a density of 104 cells in each well were seeded in a 96-well plate, and incubated in a humidified atmosphere of 5% CO2 at 37 °C for 24 h. The uncoated TPU and PL-AOT coated TPU were placed on the top of the cell layer. The parallel 3

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Simply, the needle of the catheter was provisionally removed, and a 0.4 cm section from the tip of the catheter were cut off and rinsed in 75% ethanol for 15 min. For uncoated samples, the 0.4 cm piece and the remaining catheter portion were re-assembled back onto the original needle. For coated samples, the 0.4 cm sections were immersed in PLAOT complex ethanol solution before being assembled back onto the needle (Supplementary Scheme S3A).

experiment without the samples was conducted as a blank control. After 24 h of incubation at 37 °C, the culture medium was removed, followed by the addition 90 μL of culture medium and 10 μL of CCK-8 solution into the wells. After 2 h of incubation, the obtained formazan crystals to measure their optical absorbance at a test wavelength of 450 nm using a microplate reader (TECAN SUNRISE, Swiss). The results were expressed as percentages relative to the control experiment. Cell viability was calculated using equation (2):

cell viability =

OD 450 nm (samples ) × 100 OD450nm (controls )

2.9.1. Host response to sterile subcutaneous implantation of PL-AOT coated catheter The investigations of the tissue compatibility were carried out by histopathological slices for studying the host reaction of foreign bodies in vivo. All mice were administered inhalational anesthesia with isoflurane for implantation. The back area of the mice was shaved clearly and disinfected strictly. Based on the size of the sample, a 0.2 cm incision was made on both sides of the back using surgical scissors. The PL-AOT coated catheter and uncoated catheter were implanted in the same mouse for comparison. The reassembled catheters without bacterial inoculation were gradually implanted through the incision. As soon as the 0.4 cm catheter segment was confirmed to be entirely inside subcutaneous tissue, the needle was removed while the pusher was pushed slightly inward. This dislodged the catheter segment with the length of 0.4 cm into the subcutaneous tissue, such that once the pusher was removed, the only thing that remained inside the mouse muscle was the implanted 0.4 cm catheter piece (Supplementary Scheme S3B). The mice were euthanized after implantation for 5 days, observed for any significant changes in the site of implantation of coated and uncoated catheter. To visualize infection and inflammation around the implants, histological analysis of tissue sections was performed. Simply, the fresh tissue of implant sites was first fixed in 4% of PFA for 24 h and then dehydrated in 10% and 30% sucrose solution for 24 h. The tissues after frozen section were stained with H&E stain kit following the manufacturer's protocol and visualized using fluorescence microscope (Leica DM2500).

(2)

The hemolytic activities of the uncoated and PL-AOT coated catheters against erythrocyte cells were studied via a method established by Lim K [18]. Fresh rabbit red blood cell (RBC) was diluted to 5.0 vol% with PBS. The diluted RBC (200 µL) was placed on the uncoated and PLAOT coated catheter surfaces in each well of a 96-well plate. Subsequently, the plate was incubated for 1 h at 37 °C. After incubation, the RBC solutions were centrifuged at 3000 rpm for 5 min. Aliquots (100 mL) of the supernatant from each sample were transferred to a new 96-well plate, and hemoglobin release was measured at 540 nm using the TECAN absorbance reader. In this assay, red blood cells treated with 0.2% Triton X were used as a positive control, and the red blood cells in PBS were used as a negative control. Hemolysis percentage was calculated using the equation (3):

Hemolysis(%) =

OD540 of the sample − OD540 of the negative control OD540 of the positive control -OD540 of the negative control × 100%

(3)

2.9. In vivo assay All animal experiments were conducted in accordance with the guidelines of the Animal Care and Ethics Committee of Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Two groups of twenty male BALB/C mice at 8 weeks were tested in the experiments. One group was conducted host response to sterile subcutaneous implantation of PL-AOT coated catheter. The remaining group was involved in implant-associated bacterial infection to evaluate the performance of PL-AOT coated catheter via the mice subcutaneous model (Scheme 1). In order to reduce the trauma area, thereby reducing the inflammatory response after catheter implantation, I.V. Catheter (TPU, 24G) was used as an implant. Prior to animal studies, uncoated and coated I.V. catheters were prepared under an aseptic environment.

2.9.2. Mice subcutaneous model of indwelling catheter-related infection The same implant procedure was conducted except the implanted catheters had been incubated in S. aureus suspension (1 × 106 CFU mL−1) at 37 °C for 3 h. After a 5 day implant period, the catheters were visually scored for degree of associated inflammation and the entire implant and associated peri-implant tissue was surgically removed and processed, as described above. The implants were taken out and washed in sterile PBS buffer to remove non-adhered bacteria. Subsequently, the

Scheme 1. Illustration of in vivo assay include two group, tissue compatibility assay was conducted to explore the host response to sterile subcutaneous implantation of PL-AOT coated catheter, antibacterial experiment was carried out based on a subcutaneous model of indwelling catheter-related infection. 4

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Fig. 1. The 1H NMR spectrum of PL-AOT complex in d-DMSO.

Fig. 2. Surface characterization of the PL-AOT coated TPU. (A) Water contact angle measurement. (B) ATR-FTIR spectra of (a) TPU, (b) TPU/PL, (c) TPU/AOT and (d) TPU/PL-AOT. (C) XPS survey spectra for detection of C, N, O and S elements. (D) High-resolution N 1s and S 2p XPS spectra and their peak-fitting curves of the samples.

result is an average of at least three parallel experiments. The statistical significance was assessed by analysis of variance (ANOVA), *(p < 0.05), **(p < 0.01), ***(p < 0.001).

implants were immersed in the PBS buffer and ultrasonicated for 2 min to release the adherent bacteria into PBS solution. Then the bacteria suspension was diluted and plated for colony counts at 37 °C for 24 h. 2.10. Statistical analysis All data are presented as mean ± standard deviation (SD). Each 5

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3. Results and discussion

AOT complex coated TPU surface, increased from 2% to 5.12% and 0.2% to 2.19%, respectively. As shown in Fig. 2D, the uncoated TPU shows a peak at 399.7 eV, corresponding to its own urethane peak. However, PL-AOT coated TPU shows a new peak at 401.6 eV, which indicates the protonated amino group from PL of the complex. As shown in the high-resolution S 2p XPS spectra, PL-AOT coated TPU shows a peak at 167 eV remarkably, which corresponds to the sulfonate group of AOT. The XPS results confirmed the successful coating of PLAOT onto the TPU surface [43].

3.1. Characterization of PL-AOT complex The structure of the PL-AOT complex was identified by 1H NMR at ambient temperature (Fig. 1). Based on the proton peak area of secondary amines in PL, the area of each proton peak on PL is proportional to the area of proton peak on AOT, which conforms to the predicted structure. For example, the ratio of the peak area of HA in PL to HJ in AOT is 1/2. It is confirmed that the PL-AOT complex was composed of a stoichiometric ratio of PL and AOT. Stoichiometric polyelectrolytesurfactant complexes can be viewed as a new type of comb-shaped polymer, in which every polymer chain unit has an electrostatically bound “side chain” [36]. Both electrostatic interactions between charged components as well as hydrophobic interactions between the polyelectrolyte backbone and the surfactant's alkyl tail are important in stabilizing the water-insoluble complex. Polypeptide-surfactant selfassemblies have received considerable interest as peptides can adopt secondary structures, such as α-helices and β-sheets, in addition to random coils [29,37–39]. Complexes of poly(L-lysine) with oppositely charged lipids have been reported to adopt lamellar structures in the solid state, consisting of layers of polypeptide chains in the β-sheet conformation sandwiched between lipid bilayers and Poly(L-lysine) also adopt in the protonated form in the solid state [40,41]. The presence of electrostatically bound “side chains” does not preclude ordered secondary structures of the polypeptide chains, however, the ordered conformations of the polypeptide chains in the complexes are considerably more susceptible to changes in environment [28].

3.3. Antibacterial activity of PL-AOT coated TPU films For decades, bacterial colonization and biofilm formation on biomedical devices have been a challenge to the medical community [9,44]. In order to reduce the occurrence of biofilm, the indwelling biomedical devices must have the antibacterial properties. The JIS Z 2801 standard was adopted to test the antibacterial activity of PL-AOT coated films via colony count. A large number of live bacteria could be seen on the plate for the uncoated TPU films and the growth of the bacteria increased by 100 times in 24 h. In contrast, no bacteria were observed on the plate of the PL-AOT coated TPU films, indicating better antibacterial activity of the PL-AOT coating (Fig. 3A). The bactericidal activity value (R) of the PL-AOT coated TPU is far more than 2, which indicated the antibacterial ratio is higher than 99.999% for S. aureus. The morphology of bacteria on the sample surfaces were observed by SEM. As shown in Fig. 3B, lots of alive bacterial clusters with intact bacterial cell were observed on the uncoated TPU films, in contrast, the adherent bacteria on the surfaces coated with PL-AOT were obviously reduced and dead bacteria evaluated from lesions and distortions of the cell membrane of microorganisms could be seen. The above results showed that the PL-AOT complex coating has remarkable antibacterial activity for S. aureus. The bacterial adhesion and antibacterial activity of E. coil were also conducted. The coating is also bactericidal against E. coli, which demonstrates that the coating has a broad-spectrum antibacterial activity (Supplementary Fig. S2)

3.2. Surface characterization of PL-AOT coated TPU films The surface wettability of TPU/PL-AOT and control samples was presented in Fig. 2A. TPU is a hydrophobic material with a contact angle of 98.8°, while the contact angle for TPU/PL-AOT was decreased to 40.4°, indicating the enhancement of hydrophilicity of TPU surface after coated with PL-AOT. The presence of amine and sulfonic acid functional groups on the coating may result in the drastic decrease of the water contact angle. TPU/PL and TPU/AOT did not change the contact angle of TPU after washed with water of 37 °C. As PL and AOT dissolve in water, it is easily washed away in the subsequent washing process. It also proves that the PL-AOT coating is very stable when implanted in the body. Most medical catheters are usually made of flexible, durable and hydrophobic polymer materials, such as polyethylene (PE), silicon rubber and latex. In order to examine whether the PL-AOT complex could be coated onto the surfaces of a wide range of substrates, different materials such as polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC), polyethylene terephthalate (PET), SEBS elastomer, glass and Si sheets were tested. As we known that crystal violet (CV) is a positive charge dye that can be effectively combined with the PL-AOT complex. Therefore, it is possible to verify the universality of the coating by comparing the color changes of the coated and the uncoated substrates. The CV stained PL-AOT coated materials shows a deeper color than CV stained uncoated materials, demonstrating that the coating could be formed on various materials. (Supplementary Fig. S1). The FTIR measurement shows that the PL-AOT coated film exhibit the peak at 1046 cm−1 (Fig. 2B), which is representative of sulfonic acid group bands [42]. It indicated that PL-AOT was successfully coated onto the surface of TPU film. Since the complex contains protonated amino group (-NH3+) and sulfonate group (-SO3-) in its structure, the existence of nitrogen and sulfur could be revealed through XPS. The XPS further revealed the significant changes in the atomic concentrations (C, N, O, and S) presented on each TPU surface before and after coating. The numeric values of the surface atomic concentrations in each sample were listed in Supplementary Table S1. Compared to the uncoated TPU surface, the atomic concentration of N and S of the PL-

3.4. Antibacterial activity of PL-AOT coated catheter under static and dynamic flow condition The antibacterial properties of the PL-AOT coated catheters were assessed for S. aureus growth under static and dynamic flow conditions. As shown in Fig. 4A, the control samples presented significant bacterial growth after 8 h, while the OD540 of the PL-AOT coated catheter had been maintained at the initial value own to the initial bacterial suspension were killed by the coating, indicating that the antibacterial activity of PL-AOT coated catheter is better. The same results were obtained via colony plate count (Fig. 4B). In order to simulate the flow aspects of the catheters in practical applications and to ensure that the coating remains antibacterial property in a dynamic flowing environment, a microfluidic cultivation system was established. From Fig. 5A, a few bacteria and biofilms were detected on the inner surfaces of uncoated catheters at 24 h. However, bacteria inside catheter lumen grew rapidly and biofilms filled the entire lumen surface of unmodified catheters after 24 h of continuous cultivation. On the contrary, the PL-AOT coated catheters demonstrated better antibacterial ability. No obvious biofilms were found on the coated catheters after 48 h of continuous cultivation. The better antibacterial ability of catheters was also confirmed by spread colony counting experiment and SEM (Fig. 5B, C). Only a few live bacteria were detected on the lumen surfaces of the coated catheter during the entire period of cultivation. Total live bacteria for unmodified catheters grown rapidly after 48 h cultivation and the lumen of catheters were fully blocked by biofilms. The number of bacteria of uncoated catheters at 24 h was 40 times as that of coated catheters and up to 100 times at 48 h. The highly bactericidal surface of the PL-AOT coated catheter has eliminated the bacteria before they colonize on the surface, thus 6

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Fig. 3. Antibacterial activity of uncoated and PL-AOT coated TPU films (A) The colony count conducted in accordance with the JIS Z 2801 standards. The covered S. aureus suspension on the uncoated TPU films were diluted with PBS 50 times after incubated at 37 °C for 24 h, while the TPU/PL-AOT is directly spread without dilution. (B) Representative SEM images of S. aureus attachment to samples: (a) TPU, (b) TPU/PL-AOT. Samples were incubated in growth medium containing 106 bacterial cells mL−1 for 24 h. Green arrows indicate intact bacterial cells, and red arrows indicate lesions and distortions on the cell membrane of microorganisms.

implanted indwelling catheter, it can be used from a few days to several weeks. It is of great significance to preserve the long-term antibacterial stability of the coated catheters during its application, which could reduce the pain of the patient and greatly decrease the rate of bacterial infection when the catheter is implanted into the body. Therefore, the long-term antibacterial activity of the catheter in the human environment was simulated in vitro. The samples were immersed in 0.9% NaCl aqueous solution for more than twenty days to assess the long-term stability of PL-AOT coated catheters, which is close to the body environment in vivo. The antibacterial properties of the catheters with different immersion days were evaluated. OD540 measurement of the PL-AOT coated catheters and control samples at 18 h indicated that the PL-AOT coated catheters retained their antibacterial activity in 0.9% NaCl aqueous solution. It illustrated that there is a strong electrostatic interaction between PL and AOT. Therefore, the 0.9% NaCl aqueous solution has no effect on the stability of PL-AOT complex (Fig. 6A). Similarly, the coating also has good stability in the urine (Fig. 6B). Numerous of the long alkyl chain of AOT anchoring onto the hydrophobic surface via hydrophobic or van der Waals interactions when immersed the TPU in the PL-AOT ethanol solution, thereby enabling the formation of a stable coating. As is reported, the pH at the surface near bacteria was between pH 5 and pH 5.5 [45,46], therefore, the protonated PL (pKa ≈ 9.9) moieties in the PL-AOT complex will be protonated deeply at pH values lower than the pKa which lead to the partial cleavage of the ionic bonds in the complex [36,47]. The extract solution was collected after leaching with water in the PL-AOT coated catheter for 24 h to checked the amount of released PL via HPLC. The absorption peak at 6.447 which is attributed to the characteristic absorption peak of PL, was below the detection limit of the equipment (Supplementary Fig.S3). Therefore, we deduced that the release of the partial AOT ion resulted in the exposure of the amino group in the PL-AOT complex to the bacterial environment. The amino group on the PL-AOT complex has a bactericidal effect on bacteria. The antibacterial mode of the coating was speculated as contact-killing oriented bactericidal surfaces. Compared with the release-killing, contact-killing oriented bactericidal

Fig. 4. The antibacterial activity of PL-AOT coated catheters against S. aureus under static condition (A) OD540 values of the inoculant added with fresh LB medium (B) The plate for bacterial count of the S. aureus bacterial suspension incubated at 37 °C after 3 h diluted with PBS.

preventing biofilm formation and increasing the serviceable life of the catheter. 3.5. The long-term antibacterial stability and antibacterial mode of the PLAOT coated catheter Depending on the severity of illness index and the different region of 7

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Fig. 5. The antibacterial activity of PL-AOT coated catheters against S. aureus under dynamic flowing condition based on a microfluidic cultivation system (A) Images of biofilm growth inside catheters. (B) The plate for bacterial count of the coated catheter and control catheter at different time (C) Representative SEM images of S. aureus on the control catheters at 24 h (a) and 48 h (a’) and PL-AOT coated catheters at 24 h (b) and 48 h (b’). Green arrows indicate intact bacterial cells, and red arrows indicate lesions and distortions on the cell membrane of microorganisms.

surfaces is more favorable to maintaining its long-term antibacterial properties owning to the bactericide will not be continuously released into the environment. At the same time, little bactericide could be

released into the body for contact-killing oriented bactericidal surfaces that may disturb the dosage during the treatment of patients.

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Fig. 6. The long-term antibacterial stability of the uncoated and PL-AOT coated catheters. Samples immersed in 0.9% NaCl solution (A) and urine (B) at different immersion days.

Fig. 7. Cytocompatibility and hemocompatibility of the coating. (A) The cytotoxicity assay of the TPU and TPU/PL-AOT films were conducted in vitro via CCK-8 assay. (B) Hemolysis assay of PL-AOT coated and uncoated catheters against erythrocyte cells.

Fig. 8. The tissue compatibility of the coated catheter based on a sterile subcutaneous implant model. (A) The image of the tissue compatibility in visually (5 days). Images are representative of 5 different catheter samples. (B) The results of H&E staining assay (Magnification 10x. Scale bar: 200 μm; Magnification 40x. Scale bar: 50 μm).

L929 fibroblasts cell viability of the coated catheter was similar to that of the uncoated sample after 24 h of incubation, which suggested that the coating does not impart any toxic effects on the L929 fibroblasts cell (Fig. 7A). The good biocompatibility is in favor of the clinical potential of applying such coating on catheters and other medical devices. To evaluate the hemocompatibility of the uncoated and PL-AOT coated catheter, the samples were tested for hemolytic tendency against RBC. Fig. 7B showed the rate of hemolysis of the coated catheters compared to the controls. The negligible cytotoxicity were observed of

3.6. Cytocompatibility and hemocompatibility Biocompatibility is the essential characteristic of biomedical polymer materials, which is the key to the successful application of products in clinical translation. However, bactericidal coatings developed by impregnating antibiotics, metal nanoparticles, or various biocides are limited due to cumulative toxicity, drug resistance, and rapid release of biocides[48–50]. Herein, the cell cytotoxicity of the coating was evaluated by a CCK-8 assay. The CCK-8 evaluation showed that the 9

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Fig. 9. The antibacterial property of coated catheter in an implant-associated bacterial infection model. (A) The image of the inflammatory reaction in visually (5 days). Images are representative of 12 different catheter samples. (B) Inflammation scores from PL-AOT coated catheter and untreated catheter (C) Numbers of viable S. aureus recovered from the uncoated and PL-AOT coated catheters (D) The results of H&E staining assay (Magnification 10x. Scale bar: 200 μm; Magnification 40x. Scale bar: 50 μm).

subcutaneously of the mouse. Tissue compatibility is one of the evaluation indicators for biocompatibility of biomaterials. The biocompatibility of PL-AOT coated catheter was conducted in a sterile subcutaneous implant model. The catheters were implanted into the subcutaneous tissue of the mice and then the frozen section was observed by H&E staining after five days. From Fig. 8A, there is no obvious difference and inflammation induced by the uncoated and PLAOT coated catheter on implanted or surrounding region. The above results were also confirmed from the staining of H&E assay, and the inflammatory response was analyzed via tissue images acquired using microscope. The morphology of the implant and peri-implant tissue is the same as that of the normal muscle tissue. The nucleus is evenly

the coated catheters, although the hemolytic activities were slightly higher for the coated catheters compared with the uncoated catheters (1.82 ± 0.38% for uncoated catheter; 2.81 ± 0.32% for PL-AOT coated catheter). The data was expressed as mean and standard deviation of three replicates. 3.7. In vivo assay The multifunctional surface with good biocompatibility and antibacterial activity is still a challenge to the biomaterials community [51]. To examine the clinical potential of the PL-AOT coating, the uncoated and PL-AOT coated I.V. Catheter were implanted 10

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Preparation and Application of Functional Materials, Hubei University.

distributed on the edge of the cells and no obvious toxicity and inflammatory reaction were observed, indicating that the biocompatibility of the coated indwelling catheter is better (Fig. 8B). A mouse model of implant-associated bacterial infection was developed to evaluate the antibacterial property of the PL-AOT coated catheter implants in vivo. The uncoated and the coated catheter inoculate with S. aureus were implanted into the both side of the mice back. After 5 days, the conditions of the wounds were observed (Supplementary Fig.S4). Supplementary Fig.S4 a-l (marked with the red circle) showed a serious inflammatory reaction accompanied with purulent phenomenon in the incisions with the uncoated indwelling catheter. However, the incisions with the coated implants looked different from that of the control groups, where no obvious sign of infection was observed (marked with the green circle). The mouse of k was chosen as the representative (Fig. 9A). The catheters were visually scored for degree of associated inflammation, based on a scoring system where 0 indicated no inflammation and 3 represented maximal inflammation [52]. The average inflammation score was 2.6 for unmodified catheter, in comparison that of 0.2 for PL-AOT coated catheters (Fig. 9B). All implants were retrieved and washed with PBS under ultrasonication to determine the number of viable bacteria on the implants. As shown in Fig. 9C, there were numerous S. aureus growing on the uncoated implants, but few bacteria were retrieved from the coated implants. The bacterial count for the coated implants was significantly lower than that of the control group, with a log reduction of 1.3. In addition, implants and surrounding tissues were harvested and stained with H&E for histological examination. Notably, substantial lymphatic infiltration was observed immediately adjacent to all uncoated catheter implant groups. In contrast, no significant change was happened for the muscle tissue region of PL-AOT coated catheter (Fig. 9D). It anticipated that this technology will provide an important strategy to reduce the risk of device-associated infection along with the attendant morbidity and mortality associated with these complications. The in vivo assay demonstrated the capacity of PL-AOT coated catheter to significantly limit the risk of device associated bacterial colonization and implant infection.

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4. Conclusions In summary, a water-insoluble PL-based coating (PL-AOT) with multi performances including significant bactericidal activity, notable stability, good biocompatibility has been successfully prepared and developed on the clinically used biomedical polyurethane catheter. The antibacterial coated catheters effectively inhibited the bacteria adhesion and the subsequent colonization or biofilm formation on the catheter surfaces under both static and dynamic flow conditions. The in vivo experiments proved that the PL-AOT coated catheter could significantly inhibit the inflammatory response introduced by bacterial around the implanted sites, while providing strong biocompatibility. The ease and efficacy of the PL-AOT complex preparation render this modification process applicable to a variety of medical devices for reduction of bacterial infections. The coating approach holds considerable potential as an antibacterial platform. This work would provide an efficient strategy for the rational design of safe antibacterial materials to fight biomedical device-associated infections. Acknowledgements This work is supported by Youth Innovation Promotion Association of CAS (Grant No. 2017269), Science Foundation for The Better Youth Scholars of Jilin Province (Grant No. 20170520123JH), High-Tech Research & Development Program of CAS-Wego Group, National Key Research and Development Program of China (Grant No. 2016YFC1100402), NSFC (Grant No. 51873079) and the Shandong Provincial Key Research and Development Plan, China (2017GGX20102), Ministry-of-Education Key Laboratory for the Green 11

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