Polydopamine-Assisted Immobilization of Copper Ions onto

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Polydopamine-Assisted Immobilization of Copper Ions onto Hemodialysis Membranes for Anti-microbial Jun Xing, Qiyou Wang, Tianrui He, Zhengnan Zhou, Dafu Chen, Xin Yi, Zhengao Wang, Renxian Wang, Guoxin Tan, Peng Yu, and Chengyun Ning ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00106 • Publication Date (Web): 30 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

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ACS Applied Bio Materials

Polydopamine-Assisted Immobilization of Copper Ions onto Hemodialysis Membranes for Antimicrobial Jun Xinga, f#, Qiyou Wangb#, Tianrui Hea, f#, Zhengnan Zhouc, f, Dafu Chend, Xin Yie, f, Zhengao Wanga, f, Renxian Wangd, Guoxin Tanc, Peng Yua, f*, Chengyun Ninga, f* a School of Materials Science and Engineering, South China University of Technology, Guangzhou, Guangdong, 510641, China; b Department of Spine Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, 510630, China; c School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, Guangdong, 510006, China; d Laboratory of Bone Tissue Engineering, Beijing Research institute of Traumatology and Orthopaedics, Beijing Jishuitan Hospital, Beijing, 100035, China; e School of Electro-mechanical Engineering, Guangdong University of Technology, Guangzhou, Guangdong, 510006, China f Key Laboratory of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou, Guangdong, 510006, China; KEYWORDS:

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Polyethersulfone; Polydopamine; Copper ions; Hemodialysis; Anti-microbial; Hemocompatibility

ABSTRACT:

The hemodialysis therapy is the primary treatment for the end-stage renal disease (ESRD) patients to extend their lives. To reduce the pathogenic infection during hemodialysis treatment, mussel-inspired polydopamine (PDA) was self-polymerized onto polyethersulfone (PES) membranes to immobilize copper ions, because PDA coating could firmly adhere to the PES membranes and immobilize antimicrobial metal ions with its functional groups. The immobilized copper ions through PDA can penetrate deeply into the inner surface of the pores of PES membrane. Water permeability test further indicates PDA coating can obviously enhance the hydrophilicity of PES membranes. And copper ion immobilized PES membranes showed effective antibacterial activity with a relative safe release amount of copper ions (below 1 ppm). Furthermore, hemolysis test results revealed that PDA-assisted antibacterial PES membranes can relieve red blood cells damage compared to the pure PES membranes. To sum up, PES hemodialysis membranes obtained the antibacterial ability by the PDA-assisted copper ions immobilization, which can enhance reduce the abuse of antibiotics, and is benefit the patient suffering from kidney disease.

1. INTRODUCTION

Nosocomial infection during hemodialysis therapy process is particularly dangerous for endstage renal disease (ESRD) patients,

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because of the need for long-time and frequent

hemodialysis therapy in complicate habitat of diverse pathogens.1-3 Antibiotics are the primary


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choice to control the infection during hemodialysis therapy, but the abuse of antibiotics tends to increase pathogen resistance to antibacterial threatens.4,5 Therefore, it is of great significance to construct antibacterial coating on the surface of dialysis membranes to realize local antibacterial, which can decrease the use of bacterial antibiotics, reduce the pathogen resistance, and lower the heavy pill burden. Microporous PES membrane is one of the most commonly used hemodialysis membrane materials, due to its high mechanical strength, large area, excellent thermal stability and chemical stability.6 However, the surface hydrophobicity of PES easily causes adsorption of plasma protein and platelets, leading to adhesion of bacteria and bacterial secretions, causing infection and endangering the lives of patients.7-10 There have been many research efforts to introduce various functional groups on the surface of PES to improve their hydrophilicity and further functional grafting to achieve an antibacterial performant. For example, the most common methods of radiation enable surface activation, including plasma, rays, ultraviolet, etc. 7,11-13 But this modification cannot deep into the inner surface of the pores, due to the limited irradiated depth.7 Cao et al.14 took the advantage of the sulfonic groups to immobilize silver ions on the surface of the sulfonated polyethersulfone (SPES). However, the construction of sulfonic groups on PES membranes might easily lead to chain breaking or degradation of the PES body, destroying the stability of the membrane. Mussel-inspired polydopamine (PDA), which exhibits facile and effective hydrophilization, and high adhesion property to various substrates, is efficient in immobilizing antimicrobial metal particles, because it has abundant free active functional groups, including catechol, amine and imine groups. Recent reports have shown that dopamine could self-polymerize on titanium surface to immobilize copper (II) from aqueous solution.15 On the other hand, due to the broad spectra and multichannel antibacterial properties


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compared to antibiotics, the immobilization of copper ions onto the materials is a good strategy to reduce the abuse use of antibiotics, which is the root of the antibiotics resistant bacteria. Furthermore, this method is more economic and relatively lower cytotoxicity to human body.16-19 Thus, it is a reliable and economic approach to immobilize copper ions on the surface of PES to build intrinsic antibacterial hemodialysis membrane materials to reduce the risk of infection during hemodialysis therapy. To obtain an antimicrobial PES hemodialysis membrane, we constructed a functional PDA coating on the surface of the PES membrane to immobilize copper ions. We hypothesized that PDA assisted in immobilizing copper ions on the hemodialysis membrane of copper ion functionalized PES (PES-PDA-Cu) to enhance hydrophilicity. In this study, to obtain an antimicrobial PES hemodialysis membrane, we constructed a functional PDA coating on the surface of the porous PES to immobilize copper ions. We hypothesized that PDA assisted in immobilizing copper ions on the PES hemodialysis membranes (PES-PDA-Cu) could enhance the water permeability, antibacterial and blood biocompatibility of the PES hemodialysis membranes. To test this hypothesis, the element and functional groups composition, the water permeability of the PES-PDA-Cu and the copper ions release amount were studied. Furthermore, the antibacterial performant of PES-PDA-Cu hemodialysis membranes against Escherichia coli (E. coli, Gram-negative) and Staphylococcus aureus (S. aureus, Gram-positive) was evaluated by antibacterial activity test. And hemolysis analysis was also applied to study its blood compatibility.

2. EXPERIMENTAL SECTION


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Materials. Polyethersulfone (PES, Ultrason E6020p) pellets were obtained from Badische Anilin-und-Soda-Fabrik (BASF, Beijing, China). N-methyl-2-pyrrolidone (NMP, AR), Polyvinyl Pyrrolidone (PVP, AR), dopamine chlorite (AR), copper (II) sulfate pentahydrate (CuSO4, AR) were purchased from Aladdin Industrial (Shanghai, China). Tris buffer (pH = 8.5) was provided by International Group Chemical Reagent Co. Preparation of PDA-modified PES membranes. Firstly, the liquid-liquid phase separation technique was used to prepare PES membranes.20 The casting solution was prepared by dissolving PES pellets and PVP by NMP, with the weight ratio of PES / PVP / NMP = 16:10:74. After eliminated bubbles by ultrasonic for a determined time, the casting solution was casted onto clean glass plate using a 100 µL casting knife and then immediately immersed into deionized water at 40 °C for 15 min to induce phase-separate. Then, the ice-cold deionized water was used to remove the redundant solvent and dissolution overnight. Finally, the prepared products were cut into a square shape of one 1.0 cm2 and dried in a vacuum oven at 20 °C. Next, a 2 mg mL-1 dopamine chlorite solution was prepared by using tris buffer (pH = 8.5), and the cut PES membranes were vertically immersed, and shaken at 37 °C for 24 h at 100 rpm to obtain PDA coated matrix. After the determined reaction time, the mixture was immersed into the ice-cold deionized water and was washed until the pH was ~ 6.0-7.0.21 And after immersed in deionized water for 2h, the resulting products were dried overnight in a vacuum oven (marked as PES-PDA). Along with the change of immersing time, the masses percentage of copper on modified surface also varied. Thus, the PES-PDA membranes were vertically immersed in copper sulfate solution (0.4 mol L-1) at 37 °C for 3, 6, 12, and 24h, respectively, and then washed the membranes with


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ultra-pure water three times. Finally, the copper ion-saturated membranes were dried in a vacuum oven for later analysis (marked as PES-PDA-Cux, x stands for immobilizing hours of copper ions of 3, 6, 12, and 24h, respectively.) Characterization. The pure PES, PES-PDA and PES-PDA-Cux samples were frozen by liquid nitrogen for a five seconds to obtain cross-section. A Field emission scanning electron microscope (FE-SEM, Evols 10 Carl Zeiss Company, Germany) matched with energy dispersive spectrometer (EDS, INCA 200, Oxford) was used to characterize the cross-section morphology and the distribution of element on the membrane surface. Raman spectroscopy (Lab RAM HR 800 HORIBA Jobin Yvon, France) and Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR, TY-9000, China) were used to investigate the functional moieties introduced onto PES membrane surface. Water permeability test (OCA15 Data physics, German) was adopted to show the hydrophilicity changes of PES membranes through modification process (n=4). X-ray photoelectron spectroscopy (XPS, Kratoms Axis Ultra DLD, UK) was used to characterize the surface elements and their valence state. Biotech synergy 2 Multi-Mode Microplate Reader (Anthos Late Instruments, Australia) was used to obtain the optical density. Atomic absorption spectroscopy (AAS, AA-1800H, China) was conducted to measure the content of copper ion to evaluated the toxicity. Antibacterial activity tests. The antibacterial properties of PES membranes, PES-PDA membranes, and PES-PDA-Cu membranes were quantitatively evaluated by counting the colony forming unit (CFU).22,23 The membranes were subjected to two bacteria strains of E. coli (ATCC25923) and S. aureus (ATCC25922) at an inoculum concentration of 107 cu mL-1. Before the antibacterial experiment, all the membranes before and after modification (each group consisted of four pieces) were sterilized by ultraviolet radiation for 30 min. And then 1 mL of the


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bacterial suspension was added to each well with the membranes, and incubated at 37 °C for 4 h. Each experiment was performed independently at least in duplicate. After incubation for a determined time, the membranes were gently rinsed three times with deionized water to remove unattached bacteria. The membrane was then sonicated in 10 mL of PBS (pH = 7.4) for 7 min at 25 °C to obtain a suspension of bacteria attached to the surface of the samples. The bacteria in each well were serially diluted 100 times with PBS (pH = 7.4), and 100 all each dilution was collected and then spread onto pre-solidified LB agar culture plate and incubated at 37 °C for 18 h. The antibacterial efficiency was assessed by bacteria survival ratio from Eq. (1)

=

− × 100%

(1)

Where Np and Nm were the numbers of colony units corresponding to the pure membranes and the PDA functionalized membranes, respectively. Hemocompatibility tests. Fresh rabbit blood with ethylenediaminetetraacetic (EDTA) acid disodium salt was firstly centrifuged at a speed of 1500 rpm for 10 min to separate platelet-poor plasma (PPP) and platelet-rich plasma (PRP). PPP was gently double diluted with normal saline (an optical density of 0.8 at 545 nm), and then blended with S. aureus suspension (107 cu mL-1) in the volume rate of 100:1. Afterwards, 1 mL of incubated suspension was co-cultured with samples (n = 4) at 37 °C for 4 h, and then centrifuged at 1500 rpm for 10 min. 200 all supernatants of each sample was transferred into a 96-well plate, and the optical density was measured at 545 nm by a microplate reader. The hemolysis rate (Hs) was measured from Eq. (2)


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% =

− × 100% −

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(2)

Where Ds, Dip and Nd were the optical density values of Samples, positive control, negative control, respectively. (PPP diluted with normal saline without bacteria and materials was used as a negative control, and diluted with deionized water at the same times as a positive control.) All the animal protocols were approved by the Animal Care and Use Committee of the General Hospital of the Guangzhou Military Command of PLA.

3. RESULTS AND DISCUSSION

Characterization of PDA-modified PES membranes. The surface morphologies of pure PES membranes and PES-PDA membranes was observed using SEM. Figure 1a showed that the cross-section SEM image of PES membranes was composed of finger-like micro-porous and serried micro-porous structures, which was consistent with the previously reported PES membrane morphologies.24 Then the PES membranes were immersed into dopamine chlorite solution to construct PDA functional coatings. Cross-section SEM image showed that the microporous structure of the PES was preserved after PDA functionalization (Figure 1b). And the obvious pores of the surface of the PES and PES-PDA membranes were observed in Figure 1c and Figure 1d. The multiscale porous structure of PES was the key to membrane separation. However, most commonly used surface modification methods, such as plasma spraying, radiation treatment and magnetron sputtering, were not able to deal with the inner surface of the PES membranes. Dopamine molecules can penetrate the pores and self-polymerize under alkaline conditions with air or oxygen as an oxidant.25,26 In Figure 1e, the color of the PES membranes became darker after immersing into an alkaline solution (Tris buffer, pH=8.5) of


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dopamine. The EDS area scanning of the cross-section surface of PES-PDA showed the presence of abundant nitrogen element, and the mass percentage of nitrogen was ~6.28% (Figure 1f). These results showed that the PDA coating was successfully constructed on the porous PES membranes and the modification deep into the pores of the membranes. The functional groups of the PDA functionalized PES membrane were analyzed by Raman and ATR-FTIR spectroscopy. As shown in Figure 2a and Figure 2b, there is no amino group or hydroxyl group in pure PES molecule structure, whereas PDA coating was abundant in these active groups. Compared to pure PES membrane, the PES-PDA membrane surface exhibited some noticeable changes in both Raman and ATR-FTIR spectra, indicating the construction of PDA coating on PES surface. PDA coating emit fluorescence when radiated by laser, showing a strong interference signal in Raman spectra, but as shown in Raman spectra (Figure 2a), the new peaks were still distinct at 1375 cm-1 and 1580 cm-1, representing catechol and quinone structures of PDA,27 respectively. In Figure 2b, the peaks of the FTIR spectrum of the PES-PDA membrane substrate at 1574 cm-1 was attributed to the deformation vibration of N-H.28 And the new characteristic peak appearing at 1650 cm-1 was assigned to stretching vibration of C=O compared with the PES membrane, because the catechol groups oxidize to form quinine during the dopamine self-polymerization.29,30 And there was a distinct broad adsorption peak at 3200 cm-1, which was the stretching vibrations of the N-H/O-H of dopamine.31 These changes further demonstrated the PDA was successfully coated on the surface of the porous PES membrane.


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Figure 1. Characterization of the PDA deposited PES membrane. The cross-section SEM images of (a) pure PES and (b) PES-PDA membranes; SEM images of the surface morphology of (c) pure PES and (d) PES-PDA membranes; (e) the digital images of pure PES and PES-PDA membranes; (f) EDS area scanning of the nitrogen element of cross-section of PES-PDA membranes.


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Figure 2. (a) Raman spectra and (b) ATR-FTIR spectra of pure PES membranes and the modified PES-PDA membranes. Hydrophilicity of PDA-modified PES membranes. The water permeability test was used to examine the effect of PDA coatings on the hydrophilicity of the PES membrane. Water dropped on porous membrane surface would permeate into membrane and finally disappear, and acting differently on hydrophilic and hydrophobic surfaces. As hydrophobic materials were repulsive to water, the water drop on surface would be put off or even rejected. But when surface hydrophilicity improved, the water dropped on membrane surface would rapidly spread out and soaked into membranes.32,33 As shown in Figure 3, after 1 all of water was dropped onto membrane surface the water contact angle was recorded within 120 s. The droplets on the pure PES membrane surfaces slowly reduced from 102° ± 2° to 76° ± 2° within 120 s. But the initial water contact angle of PES-PDA membranes was dropped to 40° ± 3° and rapidly disappeared within 60 s, indicating the enhanced surface hydrophilicity after PDA coating. The enhanced hydrophilicity was attributed to the abundant catechol, amine and imine groups of the PDA coating. Dopamine molecules may penetrate the pores inside the substrate and attach onto the pore wall via self-polymerization reaction during the PDA modification process, so that the porous PES-PDA membranes had excellent water permeability.34


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Figure 3. The water permeability test of pure PES and PES-PDA membranes (within the dripping time of 120 s). Immobilization of copper ions onto the PES-PDA membranes. As shown in Figure.4a, after copper ions immobilization, the micro-porous morphology of PES membrane was preserved, and the EDS regionally scanning result showed that the copper ions were immobilized to both the surfaces (A1, A3) and the inner parts (A2) of the PES-PDA membranes (Figure.4b). It indicated that copper modification penetrated deeply into the inner membrane pores, through the PDA selfpolymerization. XPS analysis was used to determine copper and other elements on the PDAfunctionalized membranes. The pure PES only consisted carbon, sulfur, oxygen and hydrogen. After immersed in dopamine solution for 24 h, an amino-rich PDA coating was constructed on the PES surface, thus a new peak representing nitrogen (1s, 400 eV) was observed (Figure 4c). Furthermore, the typical BE peak of copper at 932.5 eV was also investigated, indicating that copper ions were successfully immobilized onto PES surface,35 which is consistent with the dopamine assisted copper immobilization reported in our previous work.15


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Figure 4. Characterization of copper immobilized onto PES-PDA membranes. (a) The crosssection SEM images of PES-PDA-Cu membranes; (b) the regional copper element content of PES-PDA-Cu membranes; and (c) XPS spectra of PES membranes on PDA modification (PESPDA-Cu membranes coating condition: immersing in copper sulfate solution (0.4 mol L-1) at 37 °C for 24h). The EDS area scanning was used to estimate the distribution and mass percentage of copper for different immersing times. As can be seen from the cross-sectional scan of Figures 5a-5d, the copper was successfully distributed on the pores of the PES-PDA membranes. Although the specific copper content differed regionally due to the impact of the cross-sectional morphology and membrane hole depth and other factors, the overall content of copper remained relatively high. The quantitative dates of EDS of PDA functionalized PES membranes (Figure. 5e) indicated that the contend of copper immobilized on PES-PDA membranes was getting higher and higher with the immersion time in copper sulfate solution, which means that we could


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controlled the content of copper immobilized on the modified membranes by adjusting the immersing time. However, the excessive copper release has toxicity to humans. To examine the toxicity of copper ions, the concentration of released copper ions was assessed. After immersed into 3 mL deionized water at 37 °C for 4 h, the content of copper ions of the modified membranes was assessed by atomic absorption spectroscopy (AAS). We evaluated the leaching of copper from the modified membranes at deionized water for 4 h, consistent with the standard time of hemodialysis.36 Figure 5f showed that the concentrations of copper ions were 0.243 ± 0.012, 0.246 ± 0.005, 0.281 ± 0.011 and 0.322 ± 0.021 ppm, respectively. These results indicated that the copper ion concentration was slightly increased with the extension of copper sulfate immerse time. But these were lower than the maximum contaminant limit of copper in human body (below 1 ppm).37 These results suggested that the PES-PDA-Cux membranes leaked controllable amount of copper ions into the permeate and posed limited risk to the environment and human health when used for hemodialysis therapy.


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Figure 5. The ability of copper immobilized onto PES-PDA membranes. (a-d) EDS area scanning of copper element of copper ions immobilized membranes:(a) PES-PDA-3Cu; (b) PESPDA-6Cu; (c) PES-PDA-12Cu; (d) PES-PDA-24Cu; (e) the mass percentage on PES-PDA-Cux (PES-PDA-Cux, x stand for immobilizing hours of copper ions of 3, 6, 12, and 24h); (f) copper ion releasing result of PES-PDA-Cux after immersed in deionized water for 4 h. Data were means ± SD values from four individual membrane coupons. Antibacterial properties. The number of attached viable bacteria was counted by CFU counting method to quantitatively evaluated the antibacterial properties of PES-PDA-Cux membranes


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against E. coli and S. aureus. Both E. coli and S. aureus attached to the modified PES membranes showed significantly decreased survival after incubation for 4 h at 37 °C (Figure 6a and 6c). The statistical results indicated that the bacteria adhered on PES-PDA-Cux membranes reduced by over 99% as copper ions content increasing (Figure 6b, 6d). Interestingly, PDA also exhibited antibacterial effect, research had indicated that PDA itself has an anti-inflammatory effect.38 Many pathogenic bacteria adhered on surface secrete extracellular matrix to build biofilm, offering themselves a better biochemical environment for growing and proliferation. Bacteria under biofilm protection were more resistant to immune system and antibiotics, making clinical medicine less effective. Distinctly, PDA assisted copper ions modified membrane surface inhibited bacteria from biofilm construction, thus received decent antibacterial result and relatively low concentration of released toxic copper ions.


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Figure 6. Survival ratio of E.coli (a) and (b), and S. aureus (c) and (d) after co-culture with different membranes. (a) and (c) were the representative bacterial culture plate photographs of samples from the pure and modified PES membranes. (**) represents a statistically significant difference at p < 0.01, compared with pure PES membranes. Blood toxicity. Hemolytic S. aureus rapidly damage red cells of the infected blood. To further assess the ability of modified membrane to protect blood from bacteria, S. aureus infected blood was used in hemolysis test. As shown in Figure 7, unmodified PES membranes promoted bacterial caused a hemolysis rate of 7.368 ± 1.073%, meanwhile copper immobilized PES membranes effectively suppressed red cell injury induced by bacteria, and the hemolysis rate of infected blood was below 4%.39 The result of this experiment confirmed that hydrophobic PES membrane surface promoted bacteria growth and thus accelerate hemolysis. When membrane surface immobilized with copper ions, the hemolysis effect induced by S. aureus was obviously


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reduced, indicating that the PES-PDA-Cux membranes were able to protect red cells from bacterial infection.

Figure 7 Hemolysis experiment of healthy blood and bacterial polluted blood (Blank control: healthy red cell suspension). (**) represents a statistically significant difference at p < 0.01, compared with pure PES membranes.

4. CONCLUSIONS

Antibacterial PES membranes were simply and effectively prepared by PDA-assisted copper ions immobilization. The immobilized copper ions through PDA can penetrate deeply into the inner surface of the porous PES membrane. The PES-PDA-Cu membranes showed highly effective antibacterial activity with a relative safe release amount of copper ions (below 1 ppm). When contact with bacteria infected blood, copper ions immobilized PES membranes can relieve red blood cells damage compared to pure PES membranes. It is expected to be helpful to the modification of hemodialysis membrane and rapid industrial production, and to benefit the patients suffering from kidney disease.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (C.N.); Tel.: +86 020 22236059; *E-mail: [email protected] (P.Y.); Tel.: +86 13512724709 ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Grant Nos. 51772106, 31771080, 51702104) and the Natural Science Foundation of Guangdong Province (2016A030308014), China Postdoctoral Science Foundation (Grant Nos. 2017M622686, 2017M622641). Notes The authors declare no competing financial interest. REFERENCES 1. Valderrabano, F.; Jofre, R.; Lopez-Gomez, J.M. Quality of Life in End-Stage Renal Disease Patients. Am. J. Kidney Dis. 2001, 38, 443-464. 2. Sayin, A.; Mutluay, R.; Sindel, S. Quality of Life in Hemodialysis, Peritoneal Dialysis, and Transplantation Patients. Transplant. Proc. 2007, 39, 3047-3053. 3. Khan, H. A., Baig, F. K., Mehboob, R. Nosocomial Infections: Epidemiology, Prevention, Control and Surveillance. Asian Pac. J. Trop. Biomed. 2017, 7, 478-482. 4. Melander, R. J.; & Melander, C. The Challenge of Overcoming Antibiotic Resistance: An Adjuvant Approach? ACS Infect. Dis. 2017, 3, 559-563.


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GRAPHICAL ABSTRACT


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35x15mm (300 x 300 DPI)


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Polydopamine-Assisted Immobilization of Copper Ions onto

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