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[Makoto Sasaki](https://orcid.org/0000-0002-7696-6405), Rieko Hirata, Ayano Konagai, [Mitsuhiro Ebara](https://orcid.org/0000-0002-7906-0350)

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[Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from blood](https://mdr.nims.go.jp/datasets/0b735c68-b0b5-4ec0-befc-ac2398bf69a9)

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Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodRSC AdvancesPAPEROpen Access Article. Published on 22 August 2024. Downloaded on 1/29/2025 1:55:04 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineView Journal  | View IssueElectrospun EVOaResearch Center for Macromolecules anMaterials Science (NIMS), 1-1 Namiki, TsuEBARA.Mitsuhiro@nims.go.jpbGraduate School of Pure and Applied SciencTsukuba, Ibaraki, 305-8577, JapancResearch and Development Division, KurehIwaki, Fukushima, 974-8686, Japan† Electronic supplementary informahttps://doi.org/10.1039/d4ra04501gCite this: RSC Adv., 2024, 14, 26596Received 20th June 2024Accepted 10th August 2024DOI: 10.1039/d4ra04501grsc.li/rsc-advances26596 | RSC Adv., 2024, 14, 26596–H/AST-120 hybrid nanofibermembranes for removal of indoxyl sulfate fromblood†Makoto Sasaki, ab Rieko Hirata,c Ayano Konagaic and Mitsuhiro Ebara *abNanofibers containing activated carbon using poly(ethylene-co-vinyl alcohol) (EVOH) were prepared toremove indoxyl sulfate (IS) from the blood. IS is a urinary toxin that is highly toxic and triggers theprogression of chronic kidney disease (CKD). Here, nanofibers containing activated carbon (AST-120),which has been used practically as an adsorbent for indole (a precursor of IS), were fabricated viaelectrospinning for the adsorption and removal of IS from the blood. EVOH containing different ethyleneratios was used as the nanofiber material; moreover, the effect of the ethylene ratio on variousproperties of the nanofibers, such as surface wettability and the IS adsorption rate, was investigated. Asa result, EVOH/AST-120 nanofibers comprising EVOH with a low ethylene ratio exhibited faster ISadsorption behavior. This adsorption behavior agreed well with the pseudo-second-order model,suggesting that the diffusion of IS into the nanofibers is the rate-limiting step of the process ofadsorption. Furthermore, the nanofibers successfully reduced the IS concentration in the blood undercirculating conditions. Therefore, these EVOH/AST-120 nanofibers are expected to greatly improve theprognosis of patients with CKD when used in combination with the current hemodialysis therapy as anIS-adsorbing filter.1 IntroductionChronic kidney disease (CKD) is a condition in which theltration function of the kidneys declines, accompanied by theaccumulation of uremic toxins, electrolytes, and water in theblood because of a lack of excretion, causing various compli-cations. Approximately 90 substances have been reported asuremic toxins, which are classied into the three followingcategories: small water-soluble molecules, medium-sizedmolecules, and protein-bound molecules.1 During hemodial-ysis, which is the main treatment for CKD, uremic toxins areremoved via their transfer from the blood to the dialysate usingthe principles of diffusion and ltration. In this process, uremictoxins are transferred through holes located on the surface ofthe dialysis membrane, which have a diameter of a few nano-meters.2 Thus, small water-soluble molecules, such as urea, canbe efficiently removed during hemodialysis, whereas medium-sized molecules, such as b2-microglobulin and protein-boundd Biomaterials, National Institute forkuba, Ibaraki, 305-0044, Japan. E-mail:es, University of Tsukuba, 1-1-1 Tennodai,a Corporation, 16 Ochiai, Nishiki-machi,tion (ESI) available. See DOI:26603molecules (e.g., indoxyl sulfate (IS) and p-cresyl sulfate) areremoved less efficiently.3,4 Among these uremic toxins, IS ishighly toxic, as it triggers the progression of CKD by causingbrosis in the kidneys5,6 and increases the risk of cardiovasculardisease,7,8 which render its removal highly necessary. However,the clearance of IS via hemodialysis is very low, around 25–30mL min−1, which is only 10% of the clearance of urea.9AST-120 is a form of activated carbon that is administered asan oral adsorbent to patients in the conservative phase of CKD.It is responsible for adsorbing uremic toxins in the gastroin-testinal tract, thereby suppressing their accumulation in thebody.10–12 The use of AST-120 is mainly intended to delay theonset of hemodialysis; therefore, it is nsot suitable for thetreatment of patients with CKD. Therefore, the removal of ISdirectly from the blood is expected to arrest the progression ofCKD and reduce the risk of cardiovascular disease and osteo-porosis in patients undergoing hemodialysis.To use AST-120 for the removal of IS from the blood, it isnecessary to prevent its leakage into the bloodstream and avoiddirect contact with the blood. Therefore, we need to create newmaterials containing activated carbon. We developed a methodfor incorporating AST-120 into nanobers prepared usingpolymers. Previously, we reported the preparation of severalnanobers for the removal of uremic toxins usingelectrospinning,13–15 and we expected that this technique couldbe applied in the present context. The electrospinning methodis the most common nanober-production technique and uses© 2024 The Author(s). Published by the Royal Society of Chemistryhttp://crossmark.crossref.org/dialog/?doi=10.1039/d4ra04501g&domain=pdf&date_stamp=2024-08-21http://orcid.org/0000-0002-7696-6405http://orcid.org/0000-0002-7906-0350https://doi.org/10.1039/d4ra04501ghttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d4ra04501ghttps://pubs.rsc.org/en/journals/journal/RAhttps://pubs.rsc.org/en/journals/journal/RA?issueid=RA014036Paper RSC AdvancesOpen Access Article. Published on 22 August 2024. Downloaded on 1/29/2025 1:55:04 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlineelectrostatic repulsion when a high voltage is applied to a poly-mer solution.16–22 In covering the surface of activated carbonwith polymers by electrospinning, it is necessary to carefullyconsider which polymers to use. Although a high adsorptionrate of IS is desirable for the use in hemodialysis, the adsorptionrate is expected to decrease when the surface of the activatedcarbon is covered with a polymer. We hypothesized that the useof hydrophilic polymers might help maintain high adsorptionrate: because indoxyl sulfate is a hydrophilic molecule andwould be expected to diffuse more quickly into the hydrophilicpolymer layer. To demonstrate this hypothesis, poly(ethylene-co-vinyl alcohol) (EVOH) was used here as the nanober mate-rial because the physical properties of EVOH such as hydro-philicity vary greatly according to the copolymerization ratio ofthe ethylene units to the vinyl alcohol units (ethylene ratio).23,24EVOH is also a highly blood-compatible material with a proventrack record of practical use as a material for the production ofhemodialysis membranes.25 The purpose of the present studywas to fabricate nanobers containing AST-120 and to evaluatethe effect of the ethylene ratio of EVOH on its IS adsorptionperformance and hemocompatibility.2 Experimental2.1 MaterialsCrushed AST-120 was kindly supplied by Kureha Corporation(Tokyo, Japan). The EVOH copolymer Soarnol V2504RB with25 mol% ethylene was purchased from Mitsubishi ChemicalCorporation (Tokyo, Japan). The EVOH copolymer E105A waspurchased from Kuraray Co., Ltd (Tokyo, Japan). 1,1,1,3,3,3-Hexauoro-2-propanol was purchased from Tokyo ChemicalIndustry Co., Ltd (Tokyo, Japan). Dulbecco's phosphate-buffered saline and human serum albumin were purchasedfrom Nacalai Tesque Inc. (Kyoto, Japan). 3-Indoxyl sulfatepotassium salt was purchased from Carbosynth (Berkshire, UK),and the IS assay kit was purchased from Nipro Corporation(Osaka, Japan). Moreover, 25% glutaraldehyde and ethanol(super dehydrated) were purchased from Fujilm Wako PureChemical Corporation (Osaka, Japan). Porcine blood waspurchased from Tokyo Shibaura Zoki (Tokyo, Japan) as ananimal specimen for medical device research. Whole porcineblood was collected during the slaughter and dissection of pigsfor meat. To prevent coagulation, a 3.24% sodium citrate solu-tion was added to the porcine blood and transported to thelaboratory under refrigeration. Finally, the Micro BCA™ ProteinAssay Kit was purchased from Thermo Fisher Scientic K.K.(Tokyo, Japan).2.2 Preparation of the EVOH/AST-120 nanobers viaelectrospinningEVOH with an ethylene ratio of 25 mol% (EVOH25) and44 mol% (EVOH44) was dissolved in 1,1,1,3,3,3-hexauoro-2-propanol at a concentration of 4 wt%. The dissolution wasperformed by applying ultrasonic waves overnight. CrushedAST-120 was fed into a 4 wt% EVOH solution at concentrationsof 10, 20, and 30 wt% with respect to the polymer, then© 2024 The Author(s). Published by the Royal Society of Chemistrydispersed by applying ultrasonic waves for 1 min. Subsequently,electrospinning was performed using the following conditions:applied voltage, 25 kV; feed rate, 1.0 mL h−1; distance betweenthe collectors, 15 cm; and needle size, 22G; followed by collec-tion on aluminum covering the collectors.2.3 Observation of the EVOH/AST-120 nanobersThe obtained nanober mesh was coated with a platinumsputter and observed using a eld emission scanning electronmicroscope (Hitachi SU8230). The diameters of the bers weremeasured from the captured FE-SEM images using the ImageJsoware. Elemental mapping of the nanober surface wasperformed via energy-dispersive X-ray spectroscopy (EDX) usingFE-SEM.2.4 Physical properties of the EVOH/AST-120 nanobersThe content of AST-120 in the nanobers was assessed usinga thermogravimetric differential thermal analysis (TG-DTA,Seiko Instruments TG/DTA6200). The content was calculatedfrom the residual mass of 5 mg of ber mesh when thetemperature was increased from 25 °C to 500 °C at a rate of10 °C min−1 and held at 500 °C for 30 min.The mechanical properties of the nanobers were evaluatedby examining their behavior when subjected to a tensile test(Shimadzu EZ-SX). Each nanober was cut into 1–3 cm rectan-gles, and the stress and strain weremeasured while applying theload in the longitudinal direction at 10 mm min−1.The surface wettability of the EVOH/AST-120 nanobers wasevaluated by measuring the contact angle in air using an auto-matic contact angle meter (Kyowa Interface Science DM-700). A1 mL drop of water was deposited onto nanobers that were xedon a glass slide, and the drop was photographed 3 s later, tomeasure the contact angle.Fourier transform infrared spectroscopy (FT-IR) measure-ments were performed for AST-120 powder and fabricatednanobers in absorbance mode (Shimadzu IRAffinity-1S). Thespectra were measured from 500 cm−1 to 4000 cm−1 with64 scans and a resolution of 4 cm−1.2.5 Evaluation of human serum albumin (HSA) adsorptiononto the EVOH/AST-120 nanobersHuman serum albumin (HSA) adsorption was evaluated, toassess hemocompatibility. The samples were immersed in PBSfor 1 h, then in 1 mg mL−1 HSA solution, and nally shaken at37 °C for 1 h. Aer washing the samples ve times with PBS, toremove the excess of HSA, the samples were immersed in a 5%SDS solution (solvent: 0.1 N NaOH) and shaken at 37 °C for 1 h,to detach the adsorbed HSA. Finally, the concentration of HSA inthe supernatant was measured using the BCA method, tocalculate the amount of HSA that was adsorbed onto the sample.2.6 Evaluation of platelet adhesion to the EVOH/AST-120nanobersPlatelet-rich plasma (PRP) was obtained via the centrifugation ofporcine blood. EVOH nanobers and EVOH/AST-120 nanobersRSC Adv., 2024, 14, 26596–26603 | 26597http://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d4ra04501gRSC Advances PaperOpen Access Article. Published on 22 August 2024. Downloaded on 1/29/2025 1:55:04 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinewere immersed in PBS and allowed to stand at room temperaturefor 3 h. Each sample was then immersed in PRP; aer 2 h at 37 °C, each sample was washed twice with PBS, to remove the excessof platelets. To x the platelets that had adhered to the surface ofthe sample, each sample was immersed in a 2.5% glutaraldehydesolution and allowed to stand for 2 h at room temperature.Subsequently, for dehydration, the samples were immersed in30%, 50%, 70%, and 90% ethanol solutions for 10 min each.Finally, the samples were completely dehydrated by immersingthem twice in 100% ethanol for 15 min each. Aer drying, theplatelets that had adhered to the surface of the sample wereobserved using FE-SEM.2.7 Evaluation of the IS adsorption performance of theEVOH/AST-120 nanobers in PBSAn IS solution of 5 mg dL−1 was prepared by dissolving IS inPBS. Three milliliters of the IS solution was mixed with the AST-120 powder or EVOH/AST-120 nanobers and shaken at 37 °Cfor 8 h using a constant-temperature shaker. At each elapsedtime point, the supernatant was collected and colored using anIS measurement kit; then, the IS concentration was calculatedby measuring the absorbance of the supernatant usinga microplate reader (Tecan Innite M Nano+). The adsorptioncapacity of the EVOH/AST-120 nanobers for IS was calculatedby comparing the IS concentration aer the adsorption test withits initial concentration.2.8 Evaluation of the IS adsorption performance of theEVOH/AST-120 nanobers under blood circulationPotassium indoxyl sulfate (5.89 mg) was dissolved in 100 mL ofporcine blood, to achieve a blood IS concentration of 5 mg dL−1.The EVOH25/AST-120 nanobers were set in a homemade lterholder in a tubular form, and 30 mL of blood was circulatedusing a diaphragm pump. At each elapsed time point, blood wascollected, and the IS concentration was measured.The blood samples that were collected at each elapsed timewere centrifuged, to separate the plasma, and the hemolysisratio was assessed by measuring the absorbance of the plasma.The absorbance recorded aer the addition of PBS to the bloodwas set as the 0% hemolysis rate, and the absorbance recordedaer the addition of distilled water and all the blood washemolyzed was set as the 100% hemolysis ratio.3 Results & discussion3.1 Fabrication of the EVOH/AST-120 nanobersAer fabrication via electrospinning, the EVOH/AST-120 nano-bers were observed using FE-SEM (Fig. 1). In nanobers con-taining EVOH25 (Fig. 1a–d) as well as in nanobers containingEVOH44 (Fig. 1e–h), the formation of nanobers with a uniformstructure was observed when the AST-120 content was <30 wt%.Of note, the maximum AST-120 content was 30 wt% becausespinning was not possible for any of the nanobers when theAST-120 content was 40 wt%, as the needles became clogged. Itis thought that AST-120 was present in the bulges observed inthe nanobers.26598 | RSC Adv., 2024, 14, 26596–26603The measured ber diameter distribution of EVOH25/AST-120 nanobers and EVOH44/AST-120 nanobers with 30 wt%of AST-120 are depicted in Fig. 1i and j respectively. The meanvalues of the diameter of each nanober were 680 nm and390 nm. Histograms of ber diameters, mean, maximum, andminimum values for all nanobers are shown in Fig. S1 andTable S1.† The ber diameter decreased with the AST-120content compared with the pure EVOH nanobers, and thesame phenomenon was observed for both EVOH25 andEVOH44 nanobers. This could be because the diameter of AST-120 is larger than that of the ber system; therefore, more EVOHwas used to cover the AST-120 surface, thus reducing theamount of EVOH employed to form the nanobers.3.2 Conrmation of AST-120 encapsulation using EDXThe elemental mapping of the AST-120 powder and EVOH/AST-120 nanober surface by EDX is provided in Fig. 2. On thesurface of the activated carbon AST-120 powder (Fig. 2a), X-rayenergy derived from the carbon atoms was strongly detected(Fig. 2b), whereas X-ray energy derived from the oxygen atomswas hardly detected (Fig. 2c). This was because AST-120 isa type of activated carbon and is mostly composed of carbonatoms. However, the bulge observed in the center of the EVOH/AST-120 nanobers (Fig. 2d), which appeared to be AST-120,exhibited strong X-ray energy derived from both carbon andoxygen atoms (Fig. 2e and f). This suggests that EVOH-derivedoxygen atoms were detected. Therefore, it was conrmed thatAST-120 was xed in the nanobers by covering their surfacewith EVOH. This was thought to be because the AST-120powder was dispersed in the EVOH solution during the fabri-cation of the nanobers using electrospinning, and only theEVOH component remained on the AST-120 surface because ofthe volatilization of the solvent during electrospinning. TheEVOH coating on the surface of AST-120 was expected toprevent the outow of AST-120 when used in blood, and reducehemotoxicity by preventing direct contact between AST-120and blood.3.3 Characterization of the EVOH/AST-120 nanobersFig. 3a depicts the weight changes of the AST-120 powder, EVOHnanobers, and EVOH/AST-120 nanobers during a thermog-ravimetry-differential thermal analysis (TG-DTA). The weightchanges with DTA data are also shown in Fig. S2.† AST-120 hasvery high thermal stability; therefore, even aer a temperatureincrease to 550 °C, almost no weight change was observed, witha residual mass of 97.8%. On the other hand, EVOH nanobersand EVOH/AST-120 nanobers showed 2 steps of weight loss.The rst weight loss observed at a temperature below 200 °C isconsidered due to the evaporation of water inside the materials.The second weight loss was observed from 300 °C to around450 °C. These temperatures are consistent with the thermaldecomposition temperature of EVOH, suggesting that thethermal decomposition of EVOH caused this second weightloss. The DTA curves of these nanobers in Fig. S2† show theendothermic peaks at these temperatures. These results alsosuggest that the weight loss of EVOH nanobers and EVOH/© 2024 The Author(s). Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d4ra04501gFig. 1 (a–h): FE-SEM images of the EVOH nanofibers and EVOH/AST-120 nanofibers. The ethylene ratio of EVOH was (a–d) 25 mol% and (e–h)44mol%. The content ratio of AST-120 used in the preparation was (a and e) 0 wt%, (b and f) 10 wt%, (c and g) 20 wt%, and (d and h) 30 wt%. (i andj): histogram of fiber diamter of EVOH25/AST-120 and EVOH44/AST-120 nanofibers respectively (n = 60). The content ratio of AST-120 was30 wt%.Fig. 2 Elemental mapping image of the AST-120 powder (a–c) andEVOH/AST-120 nanofibers (d–f). The red dots indicate the EDX peak ofcarbon (b and e) and the green dots indicate the EDX peak of oxygen (cand f).Paper RSC AdvancesOpen Access Article. Published on 22 August 2024. Downloaded on 1/29/2025 1:55:04 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineAST-120 nanobers are due to water evaporation and thermaldecomposition. EVOH nanobers without AST-120 exhibitedresidual masses of 2.0% and 2.4% for EVOH25 and EVOH44,respectively. In the case of the EVOH/AST-120 nanobers, theresidual mass was 31.7% and 28.3% for EVOH25/AST-120 andEVOH44/AST-120, respectively. Considering that EVOH hardlyremained because of thermal decomposition, the residual of© 2024 The Author(s). Published by the Royal Society of ChemistryEVOH/AST-120 was considered to be mostly AST-120. Therefore,it was conrmed that AST-120 was contained in the nanobersin approximately as-prepared quantities.The results of the analysis of the surface wettability of theEVOH/AST-120 nanobers using contact angle measurementsare reported in Fig. 3b. The increase in the ethylene ratio ofEVOH from 25 to 44 mol% increased the contact angle of boththe EVOH and the EVOH/AST-120 nanobers, indicating thattheir surfaces are more hydrophobic. This may be attributed toan increase in the proportion of ethylene units, which are morehydrophobic, and a decrease in the proportion of vinyl alcoholunits, which are more hydrophilic. The surface wettability of theEVOH/AST-120 nanobers was expected to affect various phys-ical properties, such as the adsorption performance of IS andblood compatibility.The stress–strain curves of the mechanical strength of thenanobers, as evaluated using tensile testing, are depicted inFig. 3c. For both EVOH and EVOH/AST-120, nanobers with anethylene ratio of 44 mol% exhibited a higher stress and strainthan did those with a ratio of 25mol%. Previous studies have alsoreported higher mechanical strength for polyethylene nanoberscompared with polyvinyl alcohol (PVA) nanobers. For example,the maximum tensile stress of PVA nanobers, the mechanicalRSC Adv., 2024, 14, 26596–26603 | 26599http://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d4ra04501gFig. 3 (a) TG-DTA curve of the AST-120 powder, EVOH nanofibers, and EVOH/AST-120 nanofibers. (b) Water contact angles on the surface ofthe EVOH nanofibers and EVOH/AST-120 nanofibers. (Mean ± SD, n = 3, *p < 0.05, **p < 0.01). (c) Stress–strain curves of EVOH nanofibers andEVOH/AST-120 nanofibers.Fig. 4 IS adsorption capacity of AST-120 in the powder state (blackline) and in EVOH/AST-120 nanofibers (blue and orange line). (Mean ±SD, n = 3).RSC Advances PaperOpen Access Article. Published on 22 August 2024. Downloaded on 1/29/2025 1:55:04 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinestrength of which was improved via the solvent volatilizationmethod, is 11.4 MPa,26 whereas the maximum tensile stress ofpolyethylene nanobers reaches 4.9 GPa.27 Polyethylene hasmorelinear chains than does PVA, which may be explain its highermechanical strength, as well as its more tightly entangled poly-mer chains and higher crystallinity. Therefore, it can be inferredthat the higher ethylene ratio observed in this nanober alsoimproves the mechanical strength because of the dominance ofthe polyethylene-derived properties.FT-IR spectra of AST-120 powder, EVOH nanobers, andEVOH/AST-120 nanobers are shown in Fig. S3.† Severalabsorbance peaks derived from EVOH were observed in at2853 cm−1 (C–H), 2926 cm−1 (C–H) and around 3300 cm−1 (O–H), which can be related to stretching vibrations, and at1329 cm−1 (C–H) and 1456 cm−1 (C–H) due to bending vibra-tions in the spectra of EVOH nanobers and EVOH/AST-120nanobers.28 The absorbance peak derived from AST-120 wasalso observed at 2317 cm-1 (C]C) in AST-120 and EVOG-AST-120 nanobers.29 In addition, numerous noisy peaks wereobserved in AST-120 and EVOH/AST-120 at 3500 to 4000 cm−1and 1300 to 1700 cm−1, which appeared to originate from thesurface functional groups of AST-120. Absorption peaks at thesame wavenumber were observed for AST-120 powder andEVOH/AST-120 nanobers. These results also conrmed thepresence of EVOH and AST-120 in the EVOH/AST-120 nano-bers. The peak observed around 3300 cm−1 in the nanoberswas considered to be derived from hydroxy groups, but its peakposition showed different values for different nanobers. Thepeak position was 3316 cm−1 for EVOH25 and EVOH44,3289 cm−1 for EVOH25/AST-120, and 3300 cm−1 for EVOH44/AST-120. EVOH25/AST-120 showed the peak shi to the lowestwavenumber among these samples. This may be due to theincreased amount of hydroxy groups in the sample withEVOH25, which resulted in stronger hydrogen bonding, whichsuppressed molecular vibrations.303.4 Evaluation of the adsorption performance of the EVOH/AST-120 nanobers for ISThe IS adsorption performance of the EVOH/AST-120 nano-bers immersed in the IS solution was evaluated. Fig. 4 reports26600 | RSC Adv., 2024, 14, 26596–26603the change in IS adsorption over time per mass of AST-120 inthe powdered or nanober state. We conrmed that the ISadsorption rate decreased when AST-120 was included in thenanobers compared with its powder state. To achieve ISadsorption onto AST-120 in the EVOH/AST-120 nanobers, itmust diffuse between the polymer chains of EVOH covering theAST-120 surface and reach AST-120. Because this diffusionprocess takes time, the adsorption rate is thought to havedecreased. In this case, EVOH25/AST-120 showed a higheradsorption rate compared with EVOH44/AST-120. As shown inFig. 3b, EVOH25/AST-120 was more hydrophilic, which may beattributed to its higher diffusion rate. Because the treatmenttime of hemodialysis is approximately 4 h, the column shouldshow a high adsorption capacity within 4 h when used as an IS-adsorbing column. Fig. 4 reveals that the IS-adsorption capacityof EVOH25/AST-120 reached a value that was not signicantlydifferent from that of the AST-120 powder aer 4 h, indicatingthat it is useful for treatment. This nal IS-adsorption capacityof AST-120 in EVOH25/AST-120 was 26.3 mg g−1, a 68-foldimprovement in adsorption performance over EVOH/zeolitenanobers within our previous study.15 The improved adsorp-tion capacity is expected to signicantly reduce the amount of© 2024 The Author(s). Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d4ra04501gFig. 5 Pseudo-first-order model and pseudo-second-order model of IS adsorption by (a) EVOH25/AST-120 nanofibers and (b) EVOH44/AST-120 nanofibers.Table 1 Comparison of the pseudo-first-order and pseudo-second-order model kinetics of the EVOH/AST-120 nanofibersFitting model Qe (mg g−1) k1 (min−1) k2 (g mg−1 min−1) R2EVOH25/AST-120 Pseudo-rst-order 24.89 0.015 — 0.9769Pseudo-second-order 29.04 — 6.110 × 10−4 0.9971EVOH44/AST-120 Pseudo-rst-order 26.55 0.004 — 0.9919Pseudo-second-order 39.08 — 7.123 × 10−5 0.9922Paper RSC AdvancesOpen Access Article. Published on 22 August 2024. Downloaded on 1/29/2025 1:55:04 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinenanobers required for treatment, which is a signicant stepforward toward practical application.The kinetics of the adsorption of IS by the EVOH/AST-120nanobers were analyzed using a pseudo-rst-order anda pseudo-second-order model,31 and the results are reported inFig. 5 and Table 1. These parameters were calculated using eqn(1) and (2), respectively:Qt = Qe(1 − e−k1t) (1)Qt ¼ k2Qe2t1þ k2Qet(2)where t is the elapsed time, Qe is the maximum adsorptioncapacity, Qt is the adsorption capacity at each time point, and k1and k2 are the rate constants. EVOH25/AST-120 exhibited higherrate constants in both the pseudo-rst-order and pseudo-second-order models. Regarding the R2 values, both EVOH25/AST-120and EVOH44/AST-120 were better tted by the pseudo-second-order model than they were by the pseudo-rst-order model.Accordingly, in recent years, many diffusion-based adsorptionprocesses have been reported to be represented by pseudo-second-order model.32,33 Therefore, this result suggests that thediffusion of IS molecules between the polymer chains of EVOHcovering the AST-120 surface is the rate-limiting step, as describedabove, and that the difference in hydrophobicity caused by theethylene ratio of EVOH plays a major role in this process.3.5 Hemocompatibility of the EVOH/AST-120 nanobersBecause albumin is an essential protein for living organisms, itsadsorption by nanobers must be suppressed. Fig. 6a shows the© 2024 The Author(s). Published by the Royal Society of Chemistryadsorption of human serum albumin (HSA) by the AST-120powder and each of the nanobers. There was no signicantdifference between the nanobers, in turn, the amount of HSAadsorbed by each type of nanober was signicantly lower thanthat observed for the AST-120 powder. When the AST-120powder was in direct contact with blood, HSA was adsorbedby activated carbon. In contrast, the surface of the EVOH/AST-120 nanobers is covered by the EVOH component; thus, AST-120 was not in direct contact with blood. This was thought tohave inhibited the adsorption of HSA.In addition, platelet adhesion must also be inhibited becauseplatelets, which are a component of the blood, cause bloodcoagulation when they adhere and aggregate on the surface of thematerial. Aer the immersion of each nanober in PRP, theadhered platelets were xed and counted based on the SEMimages, as depicted in Fig. 6b. For both the EVOH and EVOH/AST-120 nanobers, platelet adhesion was higher in nanobers withan ethylene ratio of 25 mol%. A previous study that investigatedthe relationship between the contact angle and platelet adhesionreported that a higher water contact angle at the material surfacewas associated with a higher platelet adhesion rate.34 In theEVOH/AST-120 nanobers, a larger water contact angle wasobserved for an ethylene ratio of 25 mol%. An enhanced inter-action with platelets may have resulted in increased plateletadhesion. Aer their activation, adherent platelets elongatepseudopodia and form aggregates, thus leading to blood coagu-lation.35,36 In Fig. 6c, which depicts platelets adhering ontoEVOH25/AST-120, the platelets maintained a spherical shape anddid not appear to be activated. Therefore, platelet adhesion didnot appear to cause serious hemotoxicity.RSC Adv., 2024, 14, 26596–26603 | 26601http://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d4ra04501gFig. 6 (a) Amount of human serum albumin adsorbed by the AST-120powder and nanofibers. (Mean ± SD, n = 3, *p < 0.05, **p < 0.01). (b)Amount of platelet adhesion to nanofibers, as assessed based on theFE-SEM image. (Mean ± SD, n = 10, **p < 0.01). (c) FE-SEM image ofplatelets adhering onto the EVOH25/AST-120 nanofibers.RSC Advances PaperOpen Access Article. Published on 22 August 2024. Downloaded on 1/29/2025 1:55:04 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Online3.6 IS adsorption test under blood circulation conditionsAn experiment was conducted using EVOH25/AST-120 todetermine if IS could be adsorbed under blood circulationconditions. Fig. 7 report the changes in the IS concentrationand hemolysis ratio over time in the blood aer 4 h of circula-tion of nanobers placed in a homemade lter holder inporcine blood. The IS concentration in the blood decreasedFig. 7 IS concentration (blue line) and hemolysis ratio (red line)recorded during the blood circulation test.26602 | RSC Adv., 2024, 14, 26596–26603from an initial concentration of 5.04 mg dL−1 to 3.54 mg dL−1.This nding suggests that IS can be adsorbed even when it isbound to albumin in the blood. Although the IS concentrationcould not be reduced below the standard value of 50 mg dL−1,the nanobers are intended to be used in combination withhemodialysis as an adsorption column for IS. Considering that,to some extent, IS can be removed by hemodialysis, the amountof IS removed in this experiment is considered to be sufficient.The hemolysis ratio of porcine blood increased with time,reaching 9.88% aer 4 h. It has been reported that a hemolysisratio of 10% does not indicate serious toxicity;37 here, we wereable to maintain the hemolytic rate below this threshold.4 ConclusionsEVOH/AST-120 nanobers were prepared using electrospinningfor the adsorption of IS in the blood using two types of EVOH(with ethylene ratios of 25 mol% and 44 mol%, respectively),both with amaximum AST-120 content of 30 wt%. The EVOH25/AST-120 nanobers exhibited a higher IS adsorption rate thandid their EVOH44/AST-120 counterparts, which was attributedto its greater surface hydrophilicity, as suggested by contactangle measurements. Furthermore, the adsorption behaviors twell with the pseudo-second-order model, indicating thatdiffusion was the rate-limiting step for IS adsorption. Thedifference in the diffusion rate of IS observed between thenanobers may have affected its adsorption rate. Moreover,hemocompatibility studies showed that the amount of HSAadsorption was reduced by the inclusion of AST-120 in thenanobers. Platelet adsorption was higher for EVOH25/AST-120nanobers, possibly because of the hydrophilic nature of theirsurface. Finally, the IS adsorption performance was examinedunder the following blood circulation conditions, and the ISconcentration in the blood was successfully reduced to 70.3% ofthe initial level. The use of this nanober in combination withhemodialysis as an IS adsorption lter is expected to providesufficient therapeutic effects.Data availabilityRaw data were generated at National Institute for MaterialsScience, Japan. Derived data supporting the ndings of thisstudy are available from the corresponding author on request.Author contributionsMakoto Sasaki: conceptualization, methodology, investigation,writing – original dra, visualization. Rieko Hirata: conceptu-alization, resources, writing – review & editing. Ayano Konagai:resources, writing – review & editing, supervision. MitsuhiroEbara: conceptualization, writing – review & editing, supervi-sion, project administration, funding acquisition.Conflicts of interestThere are no conicts to declare.© 2024 The Author(s). Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d4ra04501gPaper RSC AdvancesOpen Access Article. Published on 22 August 2024. Downloaded on 1/29/2025 1:55:04 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineAcknowledgementsThis work was supported by JSPS KAKENHI Grant NumberJP20H05877 and JP23KJ0260. A part of this work was supportedby “Advanced Research Infrastructure for Materials and Nano-technology in Japan (ARIM)” of the Ministry of Education,Culture, Sports, Science and Technology (MEXT).References1 R. Vanholder, R. De Smet, G. Glorieux, A. Argilés,U. Baurmeister, P. Brunet, W. Clark, G. Cohen, P. P. DeDeyn, R. Deppisch, B. Descamps-Latscha, T. Henle,A. Jörres, H. D. Lemke, Z. A. Massy, J. Passlick-Deetjen,M. Rodriguez, B. Stegmayr, P. Stenvinkel, C. Tetta,C. Wanner and W. Zidek, Kidney Int., 2003, 63, 1934–1943.2 K. Sakai, J. Membr. Sci., 1994, 96, 91–130.3 Y. Motomiya, Y. Ando, K. Haraoka, X. Sun, H. Iwamoto,T. Uchimura and I. Maruyama, Kidney Int., 2003, 64, 2244–2252.4 B. K. I. Meijers, H. De Loor, B. Bammens, K. Verbeke,Y. Vanrenterghem and P. Evenepoel, Clin. J. Am. Soc.Nephrol., 2009, 4, 1932–1938.5 H. Hou, M. Horikawa, Y. Narita, H. Jono, Y. Kakizoe,Y. Izumi, T. Kuwabara, M. Mukoyama and H. Saito, Int. J.Mol. Sci., 2023, 24, 1–14.6 T. Niwa, Nagoya J. Med. Sci., 2010, 72, 1–11.7 H. Fujii, K. Nakai and M. Fukagawa, Ther. Apher. Dial., 2011,15, 125–128.8 S. C. Hung, K. L. Kuo, C. C. Wu and D. C. Tarng, J. Am. HeartAssoc., 2017, 6, 1–8.9 S. C. Leong and T. L. Sirich, J. Toxins, 2016, 8, 358–370.10 M. Asai, S. Kumakura and M. Kikuchi, Ren. Fail., 2019, 41,47–56.11 G. Schulman, T. Berl, G. J. Beck, G. Remuzzi, E. Ritz, K. Arita,A. Kato and M. Shimizu, J. Am. Soc. Nephrol., 2015, 26, 1732–1746.12 W. C. Liu, Y. Tomino and K. C. Lu, J. Toxins, 2018, 10, 1–22.13 K. Namekawa, M. Tokoro Schreiber, T. Aoyagi and M. Ebara,Biomater. Sci., 2014, 2, 674–679.14 R. Takai, R. Kurimoto, Y. Nakagawa, Y. Kotsuchibashi,K. Namekawa and M. Ebara, J. Nanomater., 2016, 2016,1–7.15 M. Sasaki, Y. Liu and M. Ebara, Fibers, 2021, 9, 1–11.16 N. Bhardwaj and S. C. Kundu, Biotechnol. Adv., 2010, 28, 325–347.© 2024 The Author(s). Published by the Royal Society of Chemistry17 C. Bavatharani, E. Muthusankar, S. M. Wabaidur,Z. A. Alothman, K. M. Alsheetan, M. mana AL-Anazy andD. Ragupathy, Synth. Met., 2021, 271, 116609.18 T. Mukhiya, A. Muthurasu, A. P. Tiwari, K. Chhetri,S. H. Chae, H. Kim, B. Dahal, B. M. Lee and H. Y. Kim,ACS Appl. Mater. Interfaces, 2021, 13, 23732–23742.19 N. G. Siddamallappa, M. Basavaraju and C. G. Dase Gowda,Polym. Int., 2018, 67, 1511–1522.20 B. Mahesh, D. Kathyayani, G. S. Nanjundaswamy, D. ChanneGowda and R. Sridhar, Carbohydr. Polym., 2019, 212, 129–141.21 B. Mahesh, D. Kathyayani, D. Channe Gowda, A. Sionkowskaand S. Ramakrishna, Mater. Chem. Phys., 2022, 281, 125847.22 G. S. Nanjundaswamy, B. Mahesh, G. D. Channe,N. A. Chamaraja and G. Angadi, Polym. Technol. Mater.,2021, 60, 405–418.23 H. Matsuyama, K. Kobayashi, T. Maki, M. Tearamoto andH. Tsuruta, J. Appl. Polym. Sci., 2001, 82, 2583–2589.24 T. Huang, J. Song, L. Xing, X. Li and T. He, J. Membr. Sci.,2019, 579, 172–179.25 Y. Ishii, S. Yarn, H. Kanai, A. Maezawa, A. Tsuchida,R. Wakamatsu and T. Naruse, Nephron, 1996, 73, 407–412.26 A. Rianjanu, A. Kusumaatmaja, E. A. Suyono and K. Triyana,Heliyon, 2018, 4, e00592.27 J. H. Park and G. C. Rutledge, J. Mater. Sci., 2018, 53, 3049–3063.28 A. Aragón-Gutiérrez, R. Heras-Mozos, M. Gallur, D. Lópezand P. Hernández-Muñoz, Foods, 2021, 10, 1–18.29 N. Mojoudi, N. Mirghaffari, M. Soleimani, H. Shariatmadari,C. Belver and J. Bedia, Sci. Rep., 2019, 9, 1–12.30 K. Uto, Y. Liu, M. Mu, R. Yamamoto, C. Azechi,M. Tenjimbayashi, A. Kaeser, M. A. Marliac, M. M. Alam,J. Sasai and M. Ebara, Adv. Mater. Interfaces, 2024, 11, 1–10.31 Y. S. Ho and G. Mckay, Process Biochem., 1999, 34, 451–465.32 M. A. Hubbe, S. Azizian and S. Douven, BioResources, 2019,14, 7582–7626.33 T. Hoshi, M. Endo, A. Hirai, M. Suzuki and T. Aoyagi,Pharmaceutics, 2020, 12, 1–16.34 J. H. Lee and H. B. Lee, J. Biomed. Mater. Res., 1998, 41, 304–311.35 A. C. Swanepoel and E. Pretorius, Hematology, 2015, 20, 39–47.36 J. Y. Park, C. H. Gemmell and J. E. Davies, Biomaterials, 2001,22, 2671–2682.37 K. Amin and R. Dannenfelser, J. Pharm. Sci., 2006, 95, 1173–1176.RSC Adv., 2024, 14, 26596–26603 | 26603http://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g Electrospun EVOH/AST-120 hybrid nanofiber membranes for removal of indoxyl sulfate from bloodElectronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra04501g