Multifunctions of Excited Gold Nanoparticles ... - ACS Publications

Jul 8, 2016 - Kidney with Efficient Hemodialysis and Therapeutic Potential ... Department of Internal Medicine, Taipei Medical University Wan Fang Hos...
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Multi-Functions of Excited Gold Nanoparticles Decorated Artificial Kidney with Efficient Hemodialysis and Therapeutic Potential Hsiao-Chien Chen, Chung-Yi Cheng, Hsiu-Chen Lin, Hsi-Hsien Chen, Cheng-Hsien Chen, ChihPing Yang, Kai-Huei Yang, Chun-Mao Lin, Tsung-Yao Lin, Chwen-Ming Shih, and Yu-Chuan Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05905 • Publication Date (Web): 08 Jul 2016 Downloaded from http://pubs.acs.org on July 9, 2016

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Multi-Functions of Excited Gold Nanoparticles Decorated Artificial Kidney with Efficient Hemodialysis and Therapeutic Potential Hsiao-Chien Chen†,1, Chung-Yi Cheng‡,1, Hsiu-Chen Lin§,#,1, Hsi-Hsien Chen‖,¶, Cheng-Hsien

Chen▲, Chih-Ping Yang⏊, Kai-Huei Yang†, Chun-Mao Lin†, Tsung-Yao Lin†, Chwen-Ming

Shih † and Yu-Chuan Liu †,* †

Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, No. 250, Wuxing St., Taipei 11031, Taiwan



Division of Nephrology, Department of Internal Medicine, Taipei Medical University Wan Fang Hospital, 111 Hsing-Long Rd., Sec. 3, Taipei 11696, Taiwan

§

Department of Laboratory Medicine, Taipei Medical University Hospital, No. 252, Wuxing St., Taipei 11031, Taiwan

#

Department of Pediatrics, School of Medicine, College of Medicine, Taipei Medical University, No. 250, Wuxing St., Taipei 11031, Taiwan

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Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, No. 250, Wuxing St., Taipei 11031, Taiwan,



Division of Nephrology, Department of Internal Medicine, Taipei Medical University Hospital, No. 252, Wuxing St., Taipei 11031, Taiwan

⏊Department

of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical

University, 250 Wuxing St., Taipei 11031, Taiwan ▲

Graduate Institute of Medical Science, College of Medicine, Taipei Medical University, No.

250, Wuxing St., Taipei 11031, Taiwan

KEYWORDS. Multi-functions; Gold nanoparticle; Hemodialysis; Artificial kidney; Chronic kidney disease

ABSTRACT. Chronic kidney disease (CKD) is inflammation-related. Patients with chronic renal failure who undergo hemodialysis (HD) have some acute adverse effects caused by dialysisinduced oxidative stress, protein adsorption, platelet adhesion, and activation of coagulation and inflammation. Here, resonantly illuminated gold nanoparticles-modified artificial kidney (AuNPs@AK) for achieving high efficiency accompanying with therapeutic strategy for CKD during HD is proposed. The efficiency in removing uremic toxins increased obviously, especially

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in the presence of protein (closer to the real blood). The excited AuNPs@AK expressed negatively charged surface reduced the some acute adverse effects caused by dialysis-induced protein adsorption, platelet adhesion and activation of coagulation, thus avoiding thrombosis during HD. Unlike to traditional HD which provides only one function of removing uremic toxins, the solution collected from the outlet of the sample channel of excited AuNPs@AK showed an efficient free radical scavenger that could decrease dialysis-induced oxidative stress. In CKD mouse model, this antioxidative solution from excited AuNPs@AK further decreased fibronectin expression and attenuated renal fibrosis, suggesting a reduced inflammatory response. These successful in vitro and in vivo approaches suggest that resonantly illuminated AuNPs@AK in HD take multi-advantages in shortening treatment time and reducing risk of adverse effects, which promise trailblazing therapeutic strategies for CKD.

INTRODUCTION Hemodialysis (HD) has been established for treating chronic renal failure for 50 years. Patients undergoing HD may experience some acute adverse effects caused by dialysis-induced oxidative stress,1,2 protein adsorption,3 platelet adhesion,4 and activation of coagulation and inflammation.5,6 Therefore, investigations of artificial kidneys (AKs) have focused on developing hemocompatible materials, in which the major components are polylactic acid,7 poly acrylonitrile,8 polysulfone,9 polymethyl methacrylate (PMMA), or polyethersulfone (PES).10,11 Protein adsorption onto membrane surfaces can be decreased by enhancing the membrane’s hydrophilicity.11 Platelet adhesion can be inhibited by blending with polyvinylpyrrolidone (PVP).12 Immobilization of heparin on membranes prevents thrombus formation.11 Additives, including alpha-lipoic acid, vitamin E, or enzymes, suppress the formation of reactive oxygen

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species (ROS). 9,13,14 However, the functions of vitamin E and enzymes in scavenging ROS are unstable and irreversible. Additionally, conjugating linoleic acid onto membranes can further decrease protein adsorption, platelet adhesion, and coagulation times. Thus, advanced membranes with multifunctional advantages for HD urgently need to be developed. So far, there has been less research on coatings of metallic or metallic oxide nanoparticles on dialysis membranes. A polyelectrolyte multilayer with silver nanoparticles (AgNPs) exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus.15 AgNPs-treated catheters were prepared for use in HD to prevent bacterial adhesion and to act as antibacterial coatings. However, the high toxicity of AgNPs to white blood cells is concerned.16 Cell inflammation is expressed early in the progression of many chronic diseases including chronic kidney disease (CKD).17,18 CKD is a complex disease. It can be caused by a variety of different diseases and conditions. Despite the diversity of causes of CKD, the functional changes and clinical manifestations are quite similar across the spectrum. CKD can progress at different rates, depending on the underlying cause of the disease. In some cases, progression of kidney damage is relatively swift and almost always results in end-stage kidney disease. In other cases, progression is relatively slow and can be further slowed through appropriate management. Renal fibrosis is the final common manifestation of a wide variety of chronic kidney diseases. Irrespective of the initial causes, progressive CKD often results in widespread tissue scarring that leads to the complete destruction of the kidney parenchyma and end-stage renal failure.19 The pathogenesis of renal fibrosis is, in essence, a monotonous process that is characterized by an excessive accumulation and deposition of extracellular matrix (ECM) components. Over the past several years, significant progress has been made in our understanding of the cellular and molecular mechanisms of renal fibrosis, particularly the critical role local chronic inflammation

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plays in the progression of CKD including renal fibrosis and subsequent increases in the risk of mortality.20 Recently, we reported that hydrogen bonds within water molecules can be destroyed by hot electrons generating from the decay of excited gold nanoparticles (AuNPs) under resonant illumination.21,22 This water, called plasmon-activated water (PAW), improved the efficiency of removing uremic toxins. Furthermore, it produced safe HD, which was attributed to free radical scavenging and inhibition of inflammation by PAW. However, PAW with its special functions changes back to normal water over time. Also, the production and storage of large amounts of PAW are inconvenient. These disadvantages encouraged us to innovatively design a dialyzer with in situ production of PAW. Herein, we propose a significant new idea for applying AuNPs to HD. The diffusion capacity of solutes, cytotoxicity, and hemocompatibility were examined using membranes coated with AuNPs under resonant illumination. Also, fabrication of a highperformance dialyzer was achieved by coating AuNPs onto a commercial AK. Furthermore, based on the anti-inflammatory property of PAW, a CKD mouse model was generated by performing a uninephrectomy with ischemia-reperfusion injury in the contralateral kidney to assess the potential effects of PAW during the progression of CKD. EXPERIMENTAL SECTION Chemicals and Materials. Sodium chloride (99+%), creatinine (Crea), vitamin B12 (V-B12) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were purchased from Sigma-Aldrich Organics. Reagents of methylene blue (MB), H2O2 and iron(II) chloride tetrahydrate were purchased from Acros Organics. Reagent of blood urine nitrogen (BUN, 99.7%) was purchased from J. T. Baker. All of the reagents were used as received without further purification. Dialysis membrane (T1Series MWCO: 3500) used in MB-related experiments was purchased from Orange (USA).

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Commercial artificial kidney of hollow fiber dialyzers with polymethylmethacrylate (PMMA) membranes (model: B3-1.0A) was purchased from Toray Filtryzer (Japan). Commercial filter membrane of polyethersulfone (Millipore Express PLUS, 0.22 µM) was purchased from Millipore. Commercial chitosan (Ch) powders with a degree of deacetylation of 0.82 were purchased from First Chemical Works, Taiwan. All of the solutions were prepared using deionized (DI) 18.2 MΩ cm water provided from a Milli-Q system. Preparation of gold nanoparticles (AuNPs). The AuNPs in an aqueous solution was obtained from an Au sheet (purity of 0.9999) by using electrochemical and thermal reduction methods, as shown in our previous report.23 Typically, the Au electrode was cycled in a deoxygenated aqueous solution of 40 mL containing 0.1 M NaCl and 1 g L -1 Ch from -0.28 to +1.22 V vs Ag/AgCl at 500 mV s-1 for 200 scans under slight stirring. The durations at the cathodic and anodic vertices are 10 and 5 s, respectively. Immediately, without changing the electrolytes, the solution was heated from room temperature to boiling at a heating rate of 6 oC min-1 in air. After cooling the clear Au NPs-containing solution was separated from the settlement of Ch. Then the AuNPs-containing solution was placed in an ultrasonic bath for 30 min and was further centrifugalized at 3600 rpm for 2 min to remove Ch for preparing pure AuNPs in solution. Modification of dialysis membrane and filter membrane by AuNPs. 200 ppm of AuNPs solution dropped on soft dialysis membrane (T1-Series MWCO: 3500) which had been soaked in DI water for 3 h; meanwhile the other side of dialysis membrane did not contact the AuNPs solution. After 3 hour’s standing, the free AuNPs was washed out by DI water. The AuNPs coated dialysis membrane was used in the experiment of MB diffusion. The process of modification of filter membrane (polyethersulfone, PES) was prepared as follow. One side of

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PES membrane with the size of 1 × 1 cm2 was gently attached on the surface of AuNPs solution (200 ppm) for the purpose of wetting the PES membrane by AuNPs solution. To avoid absorbing excess AuNPs solution resulting in AuNPs solution penetrating to the other side of PES membrane that did not contact with AuNPs solution, the membrane was removed quickly after attaching on the surface of AuNPs solution for 3s. In this situation, the down side of PES membrane contained the AuNPs solution, but the upper side of PES membrane did not contain the AuNPs solution. Then, the one side of AuNPs-modified PES membrane through particle adsorption was obtained after drying at 40oC for 3h. Modification of AK by AuNPs. 200 ppm of AuNPs solution (100 mL) was used to fill AK from the entrance of dialysate. After 24 hour’s standing, the free AuNPs solution was extracted. The amount of adsorbed AuNPs on the AK membrane surface was evaluated from the free AuNPs solution which was washed out from AK. Diffusion test of MB based on AuNPs coated dialysis membrane. The experiment device was shown in Figure 1a. In this image, 50 mL of MB solution (0.3 mM) was placed on the left side and 50 mL of DI water was placed on the right side, where the channel within these two containers was separated by dialysis membrane. Mindfully, the side of membrane which was coated by AuNPs was contact to DI water. Under stirring at 400 rpm, the optical densities of dialyzed MB in various times at the right side were measured by ELISA at 665 nm. The test of cytotoxicity based on AuNP@PES membrane. PES and AuNPs@PES (1×1 cm2) membranes were placed in the 12-well plates for examining cytotoxicity under dark room and green light illumination. Also, the plates without PES membranes were used as the control. The RAW 264.7 macrophage cell line was grown in Dulbecco’s modified Eagle’s medium (DMEM)

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containing 10% fetal bovine serum (FBS), 100 U mL–1 penicillin, 100 μg mL–1 streptomycin, 4 mM L-glutamine, 4.5 g L–1 glucose, 1 mM sodium pyruvate, and 1.5 g L–1 sodium bicarbonate at room temperature. The test of protein adsorption to illuminated AuNPs@PES membranes. The tests of protein adsorption were performed by using PES membrane in dark room, PES membrane by illumination, AuNPs@PES membrane in dark room and AuNPs@PES membrane by illumination. Before examining, unmodified and modified membranes (1×1 cm2) were immersed in phosphate buffer (pH 7.4) for 2 h. Then these membranes were load on the 12-well plates, and 2 ml of bovine serum albumin (BSA) (2 mg mL–1) was added on these membranes, respectively; meanwhile the BSA solution were dropped on the bare side, meaning that the coated AuNPs did not contact with BSA directly. By shaken at 100 rpm under dark room or illumination for 2 h, these membranes were washed gently by PBS for three times. Finally, 500 µL of Bio-Rad Protein Assay solution were added to the membrane surfaces for measuring the optical densities at 595 nm. In addition, the evaluation of protein adsorption based on platelet poor plasma (PPP) was performed according to the above method, in which PPP was obtained after centrifuging at 4000 rpm for 15 min. The test of BSA adsorption to illuminated AuNPs. 0.5 mL of BSA (2mg/mL) was mixed with 0.5 mL of AuNPs solution (873 ppm). This mixture solution was mixed on a shaker (100 rpm) under illumination for 1 h. Then, the un-adsorbed BSA and adsorbed BSA on excited AuNPs could be separated under centrifugation at 13000 rpm. The quantity of adsorbed BSA on excited AuNPs was calculated from the free (un-adsorbed) BSA. Also, the control experiment was performed by using the same experimental condition mentioned above, but the experiment was performed in dark atmosphere.

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The test of platelet adhesion to illuminated AuNPs@PES membranes. The tests of platelet adhesion were performed by using PES membrane in dark room, PES membrane by illumination, AuNPs@PES membrane in dark room and AuNPs@PES membrane by illumination. Before examining, unmodified and modified membranes (1×1 cm2) were immersed in phosphate buffer (pH 7.4) for 2 h. Then these membranes were load on the 12-well plates, and 200 µL of platelet rich plasma (PRP) which was obtained by centrifuging blood at1000 rpm for 15 min was added on these membranes, respectively; meanwhile the PPP solution were dropped on the bare side, meaning that the coated AuNPs did not contact with PPP directly. By shaken at 100 rpm under dark room or illumination for 2 h, these membranes were washed gently by PBS for three times. Then 20 µL of cell lysis buffer were added on the membranes and reacted for 30 min. Finally, 100 µL of LDH (lactose dehydrogenate assay) Reaction Mix were added to the above solution and incubate for 30 min at room temperature. The optical density at 450 nm was measured by ELISA reader. The evaluation of thrombus formation on illuminated AuNPs@PES membranes. 200 µL of citrated blood were dropped on the dry membranes; meanwhile the citrated bloods were dropped on the bare side, meaning that the coated AuNPs did not contact with blood directly. Then, the clotting reaction was injecting 0.1 M of CaCl2 (20 µL) to those citrated bloods. After shaking gently at 100 rpm for 30 min, 5 mL of DI water was added to stop the reaction. The jelly-like thrombus on the membrane was washed by DI water further before drying in the vacuum. Finally, the weight of coagula could be obtained. Measurement of zeta potential on water. Zeta potentials of water samples from AK with and without modifications were analyzed using a Malvern Zetasizer Nano ZS zeta potential analyzer.

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Raman spectra recorded on water and their deconvolutions. In measurement prepared water (0.1 mL) was added into a cell with Ag sheet at the bottom of the cell. Raman spectra were obtained (Micro Raman spectrometer, Model UniRAM-Raman) by using a confocal microscope employing a diode laser operating at 532 nm with an output power of 1 mW on the sample. A 50x, 0.36 NA Olympus objective (with a working distance of 10 mm) was used to focus the laser light on the sample-containing Ag sheet. The laser spot size is ca. 2.5 m. A thermoelectrically cooled Andor iDus charge-coupled device (CCD) 1024 x 128 pixels operating at -40 oC was used as the detector with 1 cm-1 resolution. All spectra were calibrated with respect to silicon wafer at 520 cm-1. In measurements, a 90o geometry was used to collect the scattered radiation. A 325 notch filter was used to filter the excitation line from the collected light. The acquisition time for each measurement was 1 s. Thirty sequential measurements were collected for each sample.

Examining the removal efficiency of BUN, CREA and V-B12 based on AK under water system. In this test, the AK with and without modification by AuNPs were employed. The sample solutions were prepared by adding BUN (ca. 100 mg dL-1), CREA (ca. 20 mg dL-1), VB12 (1000 mL-1) in DI water. These solutions flowed into the AK from the top entrance and out from the bottom exit under an open system. The DI water as the dialysate flowed into the dialyzer from the bottom entrance and from the top exit. The flowing mode of these two streams is of countercurrent with various flow rates, meanwhile the types of performing dialyzer included AK in dark room, AK under LED illumination, AuNPs@AK in the dark room and AuNPs@AK under resonant illumination. The samples of residual BUN, CREA and V-B12 were collected from the bottom exit to evaluate the removal efficiency as the sample solutions flowed through the AK columns for one time. The concentration of residual BUN, CREA and V-B12 were

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analyzed as follows. BUN was tested by kinetic UV assay (cobas), while CREA was tested by picric acid jaffe method (cobas). These analyses work on Roche automated clinical chemistry analyzers modular (Modular P800, Roche Diagnostics, Indianapolis, US). V-B12 was measured by the method of competitive binding immunoenzymatic assay. Measurement of hydroxyl free radicals by electron spin resonance spectroscopy. In electron spin resonance (ESR) measurement, a Bruker EMX ESR spectrometer was employed. ESR spectra were recorded at room temperature using a quartz flat cell designed for solutions. The dead time between sample preparation and ESR analysis was exactly 1.5 min after the last addition. Conditions of ESR spectrometry were as follows: 20 mW power at 9.78 GHz, with a scan range of 100 G and a receiver gain of 6.32 × 104. Sample preparation for measuring hydroxyl free radicals. The hydroxyl free radicals were obtained by using the well-known Fenton reaction, in which ferrous iron donates an electron to hydrogen peroxide to produce the hydroxyl free radical.24,25 Because the produced hydroxyl free radicals are very unstable they are capped by spin-trapping using DMPO to form more stable complex radicals for exact detection. The sample preparation is described as follows. First, 140 μL waters from the exit of sample channel of AK which was AK in dark room, AK under resonant illumination, AuNPs@AK in dark room and illuminated AuNPs@AK with various flow rates were added in a microtube (Eppendorf). Then 20 μL PBS (10X) was added in the tube. A complex of EDTA-chelated iron(II) was prepared by mixing 0.5 mM iron(II) chloride tetrahydrate and 0.5 mM EDTA with equal volume. Subsequently, 20 μL EDTA-chelated iron(II) (0.25 mM), 10 μL H2O2 (0.2 mM) and 10 μL DMPO (2 M) were sequentially added in the tube. The final volume in the tube is 200 μL. Exact 1.5 min later from the addition of DMPO ESR

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analysis was performed. To obtain an ESR spectrum, sample was scanned for ca. 1.5 min, accumulated 8 times, and all signals were averaged. Simulated HD experiments and concentration measurements of BUN and CREA. In HD experiments, hollow fiber dialyzers with polymethylmethacrylate (PMMA) membranes (model: B3-1.0A, Toray Filtryzer, Japan) were employed. The blood (500 mL) with high concentrations of BUN (ca. 100 mg dL-1) and CREA (ca. 20 mg dL-1) flowed into the dialyzer from the top entrance and out from the bottom exit under close system. The dialysate based on saline solution (0.9 % (wt/wt) of NaCl aqueous solution) flowed into the dialyzer from the bottom entrance and from the top exit. The flowing mode of these two streams is of countercurrent. Before experiments, suitable quantities of BUN and CREA (estimation by deducting the originally normal values in blood) were added in blood to prepare blood sample with required 100 mg dL-1 BUN and 20 mg dL-1 CREA under stirring without destroying the structure of blood. The concentrations of BUN and CREA in dialyzed bloods were analyzed as follows. BUN was tested by kinetic UV assay (cobas); while Crea was tested by picric acid jaffe method (cobas). All analyses work on Roche automated clinical chemistry analyzers modular (Modular P800, Roche Diagnostics, Indianapolis, US). Examination on the biological effect of PAW using chronic kidney injured mice. All of the work involving mice was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Council of Agriculture and was approved by the Institutional Animal Care and Use Committee at the Taipei Medical University (LAC-20140292). The chronic kidney disease (CKD) model was generated in male 129S1/SvImJ mice obtained from BioLasco Taiwan (Taipei, Taiwan). CKD was induced at 8-10 weeks of age by uninephrectomy plus ischemic-reperfusion injury to the contralateral kidney as previously

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reported.26 Sham-operated mice underwent laparotomy and manual manipulation of the kidneys. One month after operation, the mice were fed a normal diet and had free access to DI water or PAW for 4 weeks. We separated the mice into 4 groups: sham-operated mice with DI water, sham-operated mice with PAW, CKD mice with DI water, CKD mice with PAW. The kidneys were harvested by laparotomy and fixed with formalin for immunohistochemistry. Immunohistochemistry. Formalin-fixed paraffin-embedded kidney specimens were sectioned into 3 to 5 μm thickness for immunohistochemistry (IHC). Slides were deparaffinized in xylene and rehydrated with 100% ethanol, 95% ethanol and water serially. We immersed the slides into antigen-retrieval buffer (10mM Tri-sodium citrate dihydrate, 0.05% Tween-20, pH 6.0) boiled at via water bath for 30 minutes. Slides were stained using an UltraVision Quanto HRP Detection kit (Thermo Scientific, Rockford, IL, USA) with specific antibodies according to the manufacturer’s instructions. The fibronectin antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Numbers of fibronectin-labeled glomeruli/mm2 of the renal cortex area were calculated from 10 microscopic views (200X) of each slide and analyzed by Student t-test. RESULTS AND DISCUSSION Performance of illuminated AuNPs@AK. Figure 1a showed the modification of commercial artificial kidney (AK) by injecting 100 ppm of the AuNPs solution with the size ca. 15 nm (maximum absorption peak at 524 nm, Figures S1 and S2) into the dialysate channel which did not contact blood and allowing this to stand for 24 h. It can be seen that the color of the dialysis fibers changed from white to pink. In addition, the AuNPs did not detach under a high flow rate, indicating that the AuNPs had strongly adsorbed onto the AK. After standing for 24 hour, the

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free AuNPs solution was extracted. The amount of free AuNPs was calculated to be ca. 138 ppm (13.8 mg of AuNPs) from the calibration cure of AuNPs solution (Fig. S3). Therefore, the quantity of adsorbed AuNPs on the AK could be calculated to be ca. 6.2 mg. To examine the efficiency of the AuNPs@AK in HD, the dialysis mode was respectively performed with three kinds of uremic toxins: blood urea nitrogen (BUN) and creatinine (CREA) as small-sized uremic toxins, and vitamin B12 (V-B12) as a medium-sized uremic toxin (Figures 1b and 1c).27 Removal efficiencies of uremic toxins increased with an increased flow rate, while flow rates of DI water and sample solution were the same. Furthermore, the mass transfer improved as the pressure of the channel for DI water flow was lower than that for the sample solution flow, meaning that the flow rate of DI water was higher than that of the sample solution. In addition, it was clearly observed that the illuminated AuNPs@AK by green light-emitting diode (LED, with a maximum centered at 530 nm) exhibited a high efficiency of removing BUN, improving 12.7% to 53.5% compared to others under various flow rates (Tables S1~S3), meaning that mass transfer improved with illumination of the AuNPs@AK (Figure 1b). The same results were also obtained for CREA and the medium-size uremic toxin (Figure 1c and 1d). Also, the similar experiment examining the efficiency of methylene blue (MB) diffusion was carried out (Figure S4a). At the beginning, 0.3 mM MB was dissolved in water on the left side of a double-cylinder glass cell, which was separated by a dialysis membrane (DM). The right side of the glass cell contained a dialysate of deionized (DI) water without MB. The DM was decorated by AuNPs, in which the AuNPs were adsorbed onto the right side of the membrane, meaning that the AuNPs did not directly contact the MB aqueous solution (Figure S5). Under illumination, the quantity of MB that diffused to the right side increased with time (Figure S4b). Also, the diffused quantity almost did not change after being replaced with an AuNPs-coated membrane without

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illumination. This demonstrated that the AuNPs adsorbed onto the membrane did not distinctly affect diffusion of the MB aqueous solution. However, the absorbance intensity obviously increased when this modified membrane was illuminated by the LED during the process. Comparing the DM (dark), DM (LED), and AuNPs@DM (dark), more than 30% of MB was detected based on the AuNPs@DM (LED) after being allowed to diffuse for 5 h. These results revealed the solute diffusion or removal could be improved after decorating AuNPs on either AK or DM and performing under resonant illumination. It had been reported that the solute diffusion coefficient was improved as it was dissolved in PAW which was obtained from the treated DI water by AuNPs. Based on this concept, in situ production PAW from excited AuNPs@AK might occur during dialysis. To examine the existence of PAW, the solution sampling from the outlet of sample channel (for blood flow) was characterized by Raman spectrum (Figure S6).21,22 Through the deconvolution of Raman spectra, about 21.6%, 21.5%, and 21.7% of the degree of the non-hydrogen-bonded structure (DNHBS) of water were obtained based on the water from the AK (dark), AK (LED), and AuNPs@AK (dark) groups, which were much closer to the 21.3% of DI water,21 indicating that hydrogen bonds within the water molecules had not been destroyed after flowing through the above systems. However, the DNHBS of water from the illuminated AuNPs@AK group was 24.9% which increased 16.9% over DI water, demonstrating that the in situ production of PAW occurred when dialysis solution flowed through the illuminated AuNPs@AK and diffused from the dialysate channel into the sample channel. In addition, the DNHBS from the same rate of 10 mL/min (24.9%) was higher than that from the same rate of 20 mL/min (23.8%). It resulted in the improved removal efficiency of illuminated AuNPs@AK to BUN decreasinig from 32.9% (the same flow rate of 10 mL/min) to 12.7% (the same flow rate of 20 mL/min), suggesting the

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level of destroying hydrogen bonds and the degree of producing PAW were flow rate-dependent. Furthermore, the DNHBS decreased further to 22.9% as flow rates of the sample and DI water were 14 and 20 mL/min, respectively. This clearly reveals that the low pressure of the dialysate channel hindered diffusion of the generated PAW from the dialysate channel to the sample channel.

Figure 1. Equipment in the HD experiments and removal efficiencies of uremic toxins based on AKs and AuNPs@AKs in a open system. (a) Photographs of the AK, AuNPs@AK, and illuminated AuNPs@AK (left to right): 1, entrance for sample (blood) injection; 2, entrance for deionized (DI) water (dialysate) injection. Removal efficiencies of (b) BUN, (c) CREA, and (d) V-B12 using different AKs with various flow rates in the dark or under LED illumination (n=3). Efficiencies were evaluated by a one-time removal of uremic toxins in an open system. Characterization of water from sample (blood) channel of illuminated AuNPs@AK.

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Figure 2a showed the function of water from sample channel in scavenging hydroxyl free radicals (•OH) by measuring the electron spin resonance (ESR). Intensities of the ESR based on waters from the AK (dark), AK (LED), and AuNPs@AK (dark) groups were close to that of DI water, meaning they were ineffective at scavenging •OH. However, the intensities decreased by about 50.2%, 30.0%, 25.7%, and 35.8% based on waters from AuNPs@AK (LED) with flow rates (sample solution/DI water) of 10/10, 10/14, 14/20, and 20/20 mL/min (Figure S7), in which the decreased intensities were consistent with the DNHBS and degree of PAW (Figure 2b). This finding discloses important information that water from the sample solution channel based on the illuminated AuNPs@AK was an effective free radical scavenger, meaning it could reduce •OH in the blood during HD. The function of scavenging •OH can possibly be attributed to the contribution of electrons which are widely known as a reducing agent. Evidence was demonstrated by measuring the water’s charge. The zeta potential of water from the illuminated AuNPs@AK’s sample channel was -25.9 mV under the same flow rates of 10 mL/min (Figure 2c). Negatively charged water significantly differed from electronically neutral waters, including DI water and waters from the AK (dark), AK (LED), and AuNPs@AK (dark) groups. This result revealed that partially active electrons existed within water molecules; meanwhile, electrons were generated from the decay of the excited AuNPs@AK under resonant illumination. Compared to the enzyme and vitamin E which were used to scavenge ROS, the mechanism of producing hot electrons from excited AuNPs is stable and endless. Also, the DNHBS and degree of scavenging •OH were expected to be associated with the value of the zeta potential of waters at various flow rates.

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Figure 2. Properties of water from an excited AuNPs@AK. (a) Spectra of hydroxyl free radicals based on exit waters which were collected from the exits of sample channels from various AKs (n=3). Bear in mind that the injected solutions at the entrance for samples were DI water. In addition, flow rates of the sample and DI water were equal (10 mL min –1). (b) Scavenging hydroxyl free radicals using waters collected from the exits of sample channels based on illuminated AuNPs@AKs with various flow rates (n=3). (c) Zeta potentials of waters collected from the exits of sample channels (n=3). Blood compatibility of excited AuNPs. Excepting the removal efficiency to uremic toxin, the test of biocompability was performed to assess the further application in clinical HD. A PES membrane, which is the major material of AKs, was used as the substrate for loading AuNPs. Bear in mind that the AuNPs were only loaded onto one side of the membrane (AuNPs@PES). The viability of RAW 264.7 cells on the membrane was evaluated by morphological criteria

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using an optical microscope (Figures 3a and 3b). Cells were cultured under illumination for 4 h; meanwhile, the side of the membrane coated with AuNPs did not directly contact the cells. The majority of RAW 264.7 cells on the AuNPs@PES (LED) membrane showed a typical circular shape that was the same as that shown in the control group without membrane treatment, indicating that cells were alive and healthy. In addition, cell viability in the AuNPs@PES (LED) and control groups did not obviously change as the time of cell culture extended to 6 h (Figure S8). This result revealed that AuNPs@PES membranes were biocompatible. Applications of AuNPs as drug carriers and as a drug for treating rheumatoid arthritis have been widely investigated, and it is known that AuNPs are biocompatible.28 Therefore, it would be expected that AuNPs-coated membranes would be non-cytotoxic. Moreover, RAW 264.7 cells did not directly contact the AuNPs. This also shows evidence that non-cytotoxic PAW was produced between the interface of the modified membrane and water under illumination during cell culture. During the HD process, protein adsorption, which first occurs when the HD membrane contacts the blood, is recognized as the main factor causing thromboses. To evaluate the hemocompatibility of AuNPs@PES membranes, protein adsorption was tested using bovine serum albumin (BSA). Figure 3c shows results of concentrations of absorbed BSA which were recorded by an enzyme-linked immunosorbent assay (ELISA) reader at 595 nm. It was observed that the quantity of adsorbed BSA on PES membranes in a dark room was close to that carried out under illumination, meaning that the green light from the LED did not affect protein adsorption. Moreover, the measured value from the test based on AuNPs@PES membranes (dark) also indicated that the adsorption capacity of BSA did not change with the AuNPs coating, because BSA was not in direct contact with the AuNPs. Interestingly, the quantity of adsorption of BSA distinctly decreased when the AuNPs@PES membrane was illuminated. The optical

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density dropped about 51.1% compared to the AuNPs@PES membrane (dark). This result can be explained by two reasons. First, it is known that proteins adsorbed onto membranes are based on hydrogen bonding, hydrophobic interactions, and electrostatic attraction.11 Also, PAW was demonstrated to provide more available sites for forming hydrogen bonds with other species. As PAW forms hydrogen bonds with proteins and membranes, it can respectively reduce the opportunity to form hydrogen bonds within proteins and membranes. In addition, AuNPs release hot electrons under resonant illumination, resulting in the production of a negative charge in the vicinity of AuNPs@PES membranes. The negatively charged AuNPs@PES membrane exhibited electrostatic repulsion to negatively charged proteins, thus decreasing the adsorption of BSA. The direct evidence was demonstrated in mixed solutions (1 mL) of AuNPs (0.5 mL, 873 ppm) and BSA (0.5 mL, 2 mg/mL) under resonant illumination and in the dark for reference. The amount of adsorbed BSA on AuNPs was quantified by assaying the free BSA in the mixture solution according to its BSA standard calibration curve (Figure. S9), in which free BSA was obtained after centrifugation at 13,000 rpm. After shaking at 100 rpm in the dark for 1 h, it was calculated that 0.51±0.09 µg of BSA was adsorbed by 1 µg of AuNPs (Figure 3d). Interestingly, the quantity of adsorbed BSA onto 1 µg AuNPs was reduced to 0.26±0.08 µg (ca. a half lower) when the experiment was performed under resonant illumination. In the experiment, the precipitated AuNPs after centrifugation were taken out and re-dispersed in a Bio-Rad Protein Assay solution. Also, the color of the AuNPs-containing solution in the dark was bluer than that under resonant illumination (inset in Figure 3d). This indicates that more BSA was adsorbed on AuNPs in the dark. These results suggest that excited AuNPs could effectively prevent the adsorption of BSA due to the electrostatic repulsion of both negative charges of excited AuNPs and BSA. Furthermore, the in vitro adsorption behavior of platelet-poor plasma (PPP) was also

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investigated (Figure 3e). With the complex compositions of plasma, compared to the PES membrane, protein adsorption decreased by more than 32.7% on an excited AuNPs@PES membrane. This demonstrates that AuNPs-coated PES membranes can efficiently prevent protein adsorption under resonant illumination. In addition to protein adsorption, platelet adhesion on DMs also leads to blood coagulation. To evaluate the blood compatibility of AuNPs@PES membranes, an in vitro test was carried out by immersion in platelet-rich plasma (PRP) (Figure 3f). The lactate dehydrogenase (LDH) assay indicated that the optical density at 490 nm based on PES membranes (dark) was 1.12±0.13 which was close to 1.21±0.16 based on PES membranes (LED) and 1.15±0.27 based on AuNPs@PES membranes (dark). In contrast, the intensity based on illuminated AuNPs@PES membranes was 0.51±0.17, which was 54.4% lower, meaning that the AuNPs@PES membrane had significantly decreased platelet adhesion when illuminated by resonant light. This result was similar to the test of protein adsorption; meanwhile charges in proteins and platelets were all negative, which formed charge repulsion to the negative charge of the excited AuNPs@PES membrane. Therefore, adsorption of proteins and adhesion of platelets to membranes were inhibited by the presence of excited AuNPs. It was reported that thromboses occurred following protein adsorption and platelet adhesion as the blood contacted the dialysis membrane surface.29 This phenomenon increases the risk of stroke.30 The above results revealed that excited AuNPs@PES membranes reduced protein adsorption and platelet adhesion, thus decreasing the risk of forming thrombi. In addition, whole blood without an anticoagulant was used to examine blood coagulation on the membrane surface after adding calcium chloride. By dropping 200 µL of human blood, about 15.8±1.45, 16.5±0.93, and 16.9±1.93 mg of coagula were formed on the PES (dark), PES (LED), and AuNPs@PES

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membranes (dark), respectively (Figure 3g). Interestingly, the weight of the formed coagulum with the excited AuNPs@PES membrane was reduced to 11.2±0.84 mg. Inhibition of coagulum formation improved by ca. 29.1%. This enhancement can perhaps be explained by the high solubility of the solute.21,22 These results revealed that the AuNPs-modified PES or AK membrane is biocompatible and can prevent protein adsorption, platelet adhesion, and thrombus formation.

Figure 3. The cytocompatibility and hemocompatibility of excited AuNPs@PES membranes. Microscopic images of the RAW 264.7 macrophage cell line growing on (a) a blank plate in the dark and (b) an excited AuNPs@PES membrane for 4 h. (c) Tests of protein adsorption based on BSA. (d) Adsorption of BSA onto AuNPs under resonant illumination and in the dark (n=3). Inset: Images of AuNPs-containing solutions under resonant illumination (right) and in the dark (left). The precipitated AuNPs were dispersed in Bio-Rad Protein Assay solutions after shaking a mixed solution of AuNPs and BSA for 1 h. (e) Tests of protein adsorption based on PPP (n=3). Tests of (f) platelet adhesion and (g) thrombus formation on the membranes (n=3). Furthermore, the in vitro mode of HD was performed using human blood in a closed system;

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meanwhile respective flow rates of blood and dialysate were kept at 14 and 20 mL/min (Figure 4a). In which, Figure 4b showed the status of one of the AuNPs-coated hollow fibers from an AuNPs@AK under illumination. Based on the above evidence (Figures 3c, 3e and 3f) that the AuNPs excited by LED illumination prevented BSA, PPP, and platelet adsorption, a multifunctional AK was proposed. The hot electrons generated from the decay of excited AuNPs could pass through the pores of the fiber, thus forming a negatively charged inner surface to inhibit adsorption of the negatively charged BSA, PPP, and platelets by charge repulsion. Treatment times for removing 50% of BUN (90 mg/dL) were ca. 2.1 h based on an illuminated AK and 1.0 h based on an illuminated AuNPs@AK (Figure 4c). Also, the times to remove 50% of CREA (24 mg/dL) were ca. 2.3 h based on an illuminated AK and 1.2 h based on an illuminated AuNPs@AK (Figure 4d). These results suggest that the use of an illuminated AuNPs@AK to replace the traditional AK could reduce treatment times by half. Moreover, enhancement of the removal efficiency in the whole-blood system was much better than that performed in the DI water system (Figures 1b and 1c). For instance, increased efficiencies of removing BUN were 97.3%, 61.9%, 45.8%, 32.3% 27.3%, and 27.3% at treatment times of 0.5, 1, 1.5, 2, 2.5, and 3 h, respectively. These data were all higher than the 14.8% of the DI water system (Figure 1b). During practical HD treatment, numerous factors could hinder the efficiency of removing uremic toxins, such as protein adsorption and platelet adhesion.

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Figure 4. Equipment in the HD experiments and removal efficiencies of BUN and CREA using saline solutions based on AKs and AuNPs@AKs in a closed system. (a) Equipment in the HD experiments: 1, entrance for blood injection at a flow rate of 14 mL/mi; 2, entrance for saline injection at a flow rate of 20 mL/min. (b) Sketch of the excited AuNPs@AK. Removal efficiencies of (c) BUN and (d) CREA based on the illuminated AK and illuminated AuNPs@AK (n=3). Performance of illuminated AuNPs@AK on HD. To confirm the effect of protein adsorption on the efficiency of removing uremic toxins, sample solutions containing BUN, CREA, V-B12, and BSA were prepared to replace the human blood, and experiments were performed using the same conditions shown in Figure 4. Figure 5a to 5c show the results of removing BUN, CREA, and V-B12 in the absence and presence of BSA. For example, average

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ca. 22.8%, 39.5%, 52.7%, and 62.6% of BUN were removed at sampling times of 15, 30, 45, and 60 min, respectively, based on the illuminated AK system without BSA. However, average BUN removal was reduced to ca. 20.4%, 31.3%, 39.8%, and 45.6% at the same sampling times in presence of BSA (4 mg/mL). The relatively decreased efficiencies for BUN were ca. 10.4%, 20.7%, 24.5%, and 27.1%, respectively (Figure 5d). The relatively decreased efficiency for BUN was defined as follows: Relatively decreased efficiency (%) = (D – DBSA)/D * 100%

(1)

For instance, at a sampling time of 15 min, the average BUN concentration decreased 22.8 mg/mL in the absence of BSA (for D) and decreased 20.4 mg/mL in the presence of BSA (for DBSA). The relatively decreased efficiency was calculated by equation (1), and this value was 10.4%. At the same time, quantities of BSA adsorbed onto the AK were ca. 216.3, 339.9, 415.1, and 467.5 mg at sampling times of 15, 30, 45, and 60 min, respectively (Figure 5g). These results confirm our hypothesis that the adsorbed BSA would hinder the removal of uremic toxins, such as BUN, CREA, and V-B12 (Figures 5a-f). The decrease in the efficiency of removing uremic toxins with an increase in the amount of adsorbed BSA was also observed in the system of the excited AuNPs@AK (Figures 5d-f). However, compared to the AuNPs-free AK system, efficiencies of removing BUN using the excited AuNPs@AK in the absence of BSA increased 36.4%, 12.8%, 11.5%, and 11.4% at sampling times of 15, 30, 45, and 60 min, respectively. Encouragingly, these increases could be further magnified to corresponding 47.4%, 22.6%, 22.5%, and 24.4% in the presence of BSA (Figure 5d), because the quantities of BSA adsorbed onto the excited AuNPs@AKs were relatively lower (Figure 5g). Figures 5e and 5f demonstrate consistent results for removing CREA and V-B12, respectively. Conclusively, compared to the AK system, efficiencies using excited AuNPs@AKs were further significantly improved in the

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presence of BSA, which was close to actual HD conditions. These results are consistent with differences in the relative removal efficiencies that were observed in ideal sample systems (Figures 1b and 1c) and in real blood sample systems (Figure 4c and 4d), in which relative removal efficiencies using excited AuNPs@AKs were higher than those using an AK in an ideal sample system. Moreover, these efficiencies further improved in the blood sample system due to the effects of BSA adsorption. These results suggest that the proposed excited AuNPs@AK could develop a maximum advantage in actual HD. In addition, the increasing removal efficiency was associated with decreasing amounts of dialysate used during the HD process, meaning it could reduce water and agent consumption.

Figure 5. Influences of protein adsorption on the efficiencies of removing uremic toxins for an AK and excited AuNPs@AK. Flow rates for injections of the sample and saline solutions were

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14 and 20 mL min–1, respectively. Removal efficiencies of (a) BUN, (b) CREA, and (c) V-B12 in the absence and presence of BSA (4 mg mL –1) (n=3). The relative removal efficiencies of (d) BUN, (e) CREA, and (f) V-B12 in the absence and presence of BSA (4 mg mL–1). (g) The amount of adsorbed BSA onto the AK and excited AuNPs@AK during hemodialysis. In vivo evaluation of illuminated AuNPs@AK in CKD Mice. Figure 2a had been demonstrated the producing PAW in sample (blood) channel of illuminated AuNPs@AK during HD could be used as an efficient free radical scavenger. Based on this feature, unlike to the traditional HD which was focused on the function of removing uremic toxins, the use of excited AuNPs@AK in HD may be developed into an alternative strategy for treating chronic diseases such as CKD which are related to local chronic inflammation. Mice were subjected to a sham operation (S) or a uninephrectomy plus ischemic-reperfusion injury to the remaining kidney (CKD-operation, or CKD), and fed DI water (S or CKD) or PAW (S-PAW or CKD-PAW). The immunohistochemical (IHC) staining showed that there was no histological difference between the kidneys of S and S-PAW mice (Figures 6a and 6b). CKD mice expressed significantly higher levels of fibronectin staining in kidneys than did CKD-PAW mice (Figures 6c and 6d). In kidneys of CKD mice, obvious fibronectin aggregation existed around renal tubules and Bowman’s capsules. Quantification of fibronectin-labeled glomeruli in the kidneys indicated that PAW consumption significantly decreased fibronectin aggregation compared to that in DI waterconsuming mice (Figure 6e). Furthermore, the thickness of Bowman’s capsules from CKD-PAW mice was thinner than CKD mice. These findings suggest that PAW from inner channel of excited AuNPs@AKs can prevent CKD mice from suffering renal fibrosis.

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Figure 6. Amelioration of renal fibrosis by PAW (water from sample channel of excited AuNPs@AK) in mice with CKD. Expression of fibronectin in renal tissues from treated mice was determined by IHC. (a) Sham-operated mice fed DI water. (b) Sham-operated mice fed PAW. (c) CKD mice fed DI water. (d) CKD mice fed PAW. Red arrows indicate Bowman’s capsules, and yellow arrows indicate renal tubules. Both DI water-treated and PAW-treated mice showed no significant increase in fibronectin in the kidney by IHC staining. CKD mice showed a significant increase of fibronectin expression in the periglomerular area and tubulointerstitium compared to CKD mice fed PAW. (e) Numbers of fibronectin-labeled glomeruli/mm2 of the renal cortex area. Data were analyzed by Student's t-test and are presented as the mean ± standard deviation. Kidneys have an intrinsic capacity for self-repair after ischemic or toxic insults. This repair

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occurs primarily through epithelial cell proliferation. Healing via focal fibrotic scarring is also seen secondary to severe parenchymal injury and serves to maintain tissue integrity. While fibrosis is a normal sequela of injury, it is thought that in CKD, the normal wound healing response fails to terminate.31 Although the exact mechanisms underlying this dysregulation are unclear, the result is an inexorable fibrogenic response and expansion of the ECM which gradually destroys the normal tissue structure.32 The aberrant ECM is composed of normal matrix proteins and proteoglycans as well as other matrix proteins normally restricted to tubular basement membranes, such as fibronectin, collagen IV, and laminin.33 Other features of renal injury include thickening of Bowman’s capsule and dilated renal tubules. Thickening of Bowman’s capsule was previously reported to be a feature of hypertensive nephrosclerosis due to the laying down of collagen by parietal epithelial cells.34 CKD mice in the present study were generated by a unilateral nephrectomy plus contralateral ischemic-reperfusion injury. CKD mice fed PAW not only demonstrated decreased fibronectin expression, but also maintained a normal thickness of Bowman’s capsule. These results indicated that PAW may have a beneficial effect of attenuating renal fibrosis in CKD by decreasing ECM deposition. CONCLUSIONS In summary, innovative application of excited AuNPs@AK in HD for safely removing uremic toxins accompanying with curing CKD was proposed for the first time. This novel AK could concurrently enhance the removal efficiency to uremic toxins and inhibit protein adsorption, platelet adhesion and thrombosis as AuNPs@AK was performed under resonant illumination. Also, it resulted in generating PAW with the property of free radical scavenger in sample channel, thus decreasing dialysis-induced oxidative stress. Furthermore, this generating PAW decreased fibronectin expression and attenuated renal fibrosis from CKD mouse model. These

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results fully demonstrated the advanced advantage of using illuminated AuNPs@AK in HD, in which combined the highly efficient and safe HD and therapy of CKD at one time. These successful approaches suggest that illuminated AuNPs@AK has emerged as a promising therapeutic strategy for CKD in clinical trials. ASSOCIATED CONTENT Supporting Information The additional Table, characteristic of AuNPs, Photograph of a AuNPs@DM, Raman spectra, ESR spectra and microscopic images of the RAW 264.7 macrophage cell line are available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author *The author to whom correspondence should be addressed. Yu-Chuan Liu ([email protected]) Author Contributions 1

Hsiao-Chien Chen, Chung-Yi Cheng and Hsiu-Chen Lin contributed equally.

ACKNOWLEDGMENT The authors thank the Ministry of Science and Technology (MOST 104-2221-E-038-012-MY3) of ROC and Taipei Medical University and Taipei Municipal Wanfang Hospital for their financial support (Taipei Medical University-Taipei Municipal Wanfang Hospital Joint Research Program; 103TMU-WFH-01-1). REFERENCES

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(1) Morena, M.; Delbosc, S.; Dupuy, A. M.; Canaud, B.; Cristol, J. P. Overproduction of Reactive Oxygen Species in End-Stage Renal Disease Patients: A Potential Component of Hemodialysis-Associated Inflammation. Hemodial. Int. 2005, 9, 37-46. (2) Ward, R. A.; McLeish, K. R. Oxidant Stress in Hemodialysis Patients: What Are The Determining Factors?. Artif. Organs 2003, 27, 230-236. (3) Mares, J.; Thongboonkerd, V.; Tuma, Z., Moravec, J.; Matejovic, M. Specific Adsorption of Some Complement Activation Proteins to Polysulfone Dialysis Membranes during Hemodialysis. Kidney Int. 2009, 76, 404-413. (4) Daugirdas, J. T.; Bernardo, A. A. Hemodialysis Effect on Platelet Count and Function and Hemodialysis-Associated Thrombocytopenia. Kidney Int. 2012, 82, 147-157. (5) Myrvang, H. Hemodialysis: Fish Oil for Hemodialysis. Nat. Rev. Nephrol. 2012, 8, 373. (6) Chen, L. P.; Chiang, C. K.; Chan, C. P.; Hung, K. Y.; Huang, C. S. Does Periodontitis Reflect Inflammation and Malnutrition Status in Hemodialysis Patients? Am. J. Kidney Dis. 2006, 47, 815-822. (7) Zhu, L.; Liu, F.; Yu, X.; Xue, L. Poly(Lactic Acid) Hemodialysis Membranes with Poly(lactic

acid)-block-poly(2-hydroxyethyl

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Preparation, Characterization, and Performance. ACS Appl. Mater. Interfaces 2015, 7, 1774817755. (8) Désormeaux, A.; Moreau, M. E.; Lepage, Y.; Chanard, J.; Adam, A. The Effect of Electronegativity and Angiotensin-Converting Enzyme Inhibition on the Kinin-Forming Capacity of Polyacrylonitrile Dialysis Membranes. Biomaterials 2008, 29, 1139-1146.

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(9) Dahe, G. J.; Teotia, R. S.; Kadam, S. S.; Bellare, J. R. The Biocompatibility and Separation Performance of Antioxidative Polysulfone/Vitamin E TPGS Composite Hollow Fiber Membranes. Biomaterials 2011, 32, 352-365. (10) Santoro, A.; Grazia, M.; Mancini, E. The Double Polymethylmethacrylate Filter (DELETE system) in the Removal of Light Chains in Chronic Dialysis Patients with Multiple Myeloma. Blood Purif. 2013, 35, 5-13. (11) Ma, L.; Qin, H.; Cheng, C.; Xia, Y.; He, C.; Nie, C.; Wang, L.; Zhao, C. Mussel-Inspired Self-Coating at Macro-Interface with Improved Biocompatibility and Bioactivity via Dopamine Grafted Heparin-Like Polymers and Heparin. J. Mater. Chem. B 2014, 2, 363-375. (12) Oo, Z. Y.; Deng, R.; Hu, M.; Ni, M.; Kandasamy, K.; Ibrahim, M. S. bin; Ying, J. Y.; Zink, D. The Performance of Primary Human Renal Cells in Hollow Fiber Bioreactors for Bioartificial Kidneys. Biomaterials 2011, 32, 8806-8815. (13) Himmelfarb, J.; Ikizler, T. A.; Ellis, C.; Wu, P.; Shintani, A.; Dalal, S.; Kaplan, M.; Chonchol, M.; Hakim, R. M. Provision of Antioxidant Therapy in Hemodialysis (PATH): A Randomized Clinical Trial. J. Am. Soc. Nephrol. 2014, 25, 623-633. (14) Mahlicli, F. Y.; Şen, Y.; Mutlu, M.; Altinkaya, S. A. Immobilization of Superoxide Dismutase/Catalase onto Polysulfone Membranes to Suppress Hemodialysis-Induced Oxidative Stress: A Comparison of Two Immobilization Methods. J. Membr. Sci. 2015, 479, 175-189. (15) Paladini, F.; Pollini, M.; Deponti, D.; Giancamillo, A. D.; Peretti, G.; Sannino, A. Effect of Silver Nanocoatings on Catheters for Haemodialysis in Terms of Cell Viability, Proliferation, Morphology and Antibacterial Activity. J. Mater. Sci.: Mater. Med. 2013, 24, 1105-1112.

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