High efficiency 3D nanofiber sponge for bilirubin removal used in hemoperfusion

Accepted Manuscript Title: High Efficiency 3D Nanofiber Sponge for Bilirubin Removal used in Hemoperfusion Authors: Zhipeng Yuan, Yansheng Li, Dan Zhao, Kexin Zhang, Fang Wang, Changtao Wang, Yongqiang Wen PII: DOI: Reference:

S0927-7765(18)30538-1 https://doi.org/10.1016/j.colsurfb.2018.08.014 COLSUB 9546

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

16-5-2018 31-7-2018 9-8-2018

Please cite this article as: Yuan Z, Li Y, Zhao D, Zhang K, Wang F, Wang C, Wen Y, High Efficiency 3D Nanofiber Sponge for Bilirubin Removal used in Hemoperfusion, Colloids and Surfaces B: Biointerfaces (2018), https://doi.org/10.1016/j.colsurfb.2018.08.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

High Efficiency 3D Nanofiber Sponge for Bilirubin Removal used in Hemoperfusion Zhipeng Yuan a, Yansheng Li a, Dan Zhao a,b, Kexin Zhang a, Fang Wang a, Changtao Wang b, Yongqiang Wen a,*

Research Center for Bioengineering & Sensing Technology, School of Chemistry and Biological Engineering,

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a

University of Science and Technology Beijing, Beijing 100083, China

Laboratory of Cosmetic (Beijing Technology and Business University), China National Light Industry, Beijing

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b Key

100048, China * for corresponding author.

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E-mail address: [email protected]

postal address: University of Science and Technology Beijing, 30th Xueyuan Road, Haidian District, Beijing

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Graphical abstract

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The total number of figures was 8.

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The total number of words was 6099.

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100083 P. R.China

3D nanofiber sponge for bilirubin removal with high efficiency and rapaid adsorption rate used in hemoperfusion

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3D nanofiber sponge with layered structure and significant high porosity

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An improved gas-foaming technique for fabricating 3D nanofiber scaffold

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Large adsorption capacity and rapid adsorption rate in plasma

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Good performances in both static adsorption and dynamic adsorption

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Highlights

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Abstract: The accumulation of bilirubin in the body could cause nervous system diseases and even endanger life in severe cases for people with liver damage or metabolic obstruction. Hemoperfusion has been considered as one of the most efficient treatments to remove extra bilirubin. Although, the current bilirubin adsorbents could adsorb the free bilirubin effectively, the albumin-bound bilirubin in plasma is hard to remove. Here, we develop a 3D nanofiber sponge fabricated by combination of electrospinning and improved gas-foaming techniques. The amino groups and BSA molecules were immobilized on the fiber surface as the affinity groups to adsorb bilirubin. The 3D nanofiber sponges have layered structure and significantly higher porosity than two-dimensional nanofiber membranes. The special 3D structure renders the sponge fully contact with the adsorbed liquid and reduces the diffusion distance of the adsorbate, thus increases the sponge's adsorption rate. The BSA immobilized nanofiber sponge showed large adsorption capacity in both aqueous solution (maximum adsorption capacity was 36.8237 mg/g) and plasma (maximum adsorption capacity was 25.2908 mg/g), rapid adsorption rate (achieved adsorption equilibrium in 60 min) and well blood compatibility.

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Key words: electrospinning; nanofiber sponge; hemoperfusion; bilirubin

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1. Introduction Bilirubin is an endotoxin which is the catabolism end product of the heme moiety[1, 2]. At the condition of liver damage or metabolic obstruction, the concentration of bilirubin in the body would increase sharply. Too much bilirubin accumulated in the body could cause nervous system diseases and even endanger life in severe cases[3, 4]. A variety of extracorporeal blood purification therapies have been widely used to remove pathogens and toxins from the bloodstream[5-7]. Among them, hemoperfusion has been considered as one of the most efficient treatments to

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remove extra bilirubin. Although, the current bilirubin adsorbents could adsorb the free bilirubin effectively, the albumin-bound bilirubin in plasma is hard to remove. An excellent hemoperfusion system requires the absorbent to

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have good blood compatibility, large adsorption capacity, high selectivity and rapid adsorption rate. Adsorbent with high adsorption efficiency in plasma has been in urgent demand.

Electrospinning is a simple and versatile method for rapid and continuous preparation of nanofibers on a large

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scale, which has been widely used in many fields including filtration[8, 9], catalysis[10, 11], sensor[12, 13], energy

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storage[14, 15] and biomedicine[16-19] uses. With their distinct small size and ultra-high specific surface area, the

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electrospun nanofiber scaffolds have great potential to be used as adsorbent for hemoperfusion[20-22]. However, the electrospinning typically produces compact two-dimensional (2D) nanofiber membrane which may restrict the fully

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contact between the adsorbent and the adsorbed liquid. Recent years, novel methods of fabricating three-dimensional

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(3D) nanofibrous scaffolds have been reported in literatures. Ding[23], Greiner[24] and Fong[25] have, respectively, reported nanofiber-assembled cellular scaffolds by combining electrospinning and freeze-drying techniques. Xie[2627] reported a modified gas-foaming approach to expand PCL electrospun nanofiber membrane in the third dimension

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used for cellular infiltration and proliferation. The 3D scaffolds have significantly higher porosity than nanofiber membrane. However, the scaffolds assembled of short fibers may not strong enough to resist the impact of flowing

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liquid and the PCL matrix does not have enough functional groups to connect functional molecules when used as adsorbent. Even though, the 3D nanofiber scaffold manufacturing technologies provided us new ideas to study the new type of hemoperfusion adsorbents.

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In this paper, we have designed and developed an innovative 3D nanofiber sponge as adsorbent for

hemoperfusion with an improved gas-foaming technique. It has been reported that bilirubin can be adsorbed by amino groups via electrostatic or hydrogen bond interactions[28,29] and can be bound to albumin at specific sites[30,31]. Therefore, amino groups and BSA were immobilized on nanofiber to absorb bilirubin. The nitrile group on the surface of electrospun PAN nanofiber was modified to carboxyl group after oxidation and protonation processes. During the 3

modification process, the small bubbles on the surface of the nanofibers made the tightly packed nanofibers separate with each other due to the generation of ammonia gas, and the nanofiber membrane became wrinkled and loose. The modification process could be considered as a pre-foaming process which helped to the subsequent foaming expansion in sodium borohydride solution. The nanofiber membrane treated with pre-foaming process were easier to expand and the expansion degree had also become more controllable. The 3D nanofiber sponge has layered structure and significantly higher porosity than two-dimensional nanofiber membrane. Special 3D structure makes the sponge

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fully contact with the adsorbed liquid and reduces the diffusion distance of the adsorbate, thus increases the sponge's adsorption rate. The adsorption performances in aqueous solution and plasma of the bilirubin affinity groups

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immobilized electrospun nanofiber sponges were investigated through adsorption kinetics, adsorption isotherms in detail. The BSA immobilized 3D electrospun nanofiber sponge could achieve adsorption equilibrium in 60 min with rapid adsorption rate while keeping high adsorption capacity. Dynamic adsorption and blood compatibility tests both

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showed that the nanofiber sponges are suitable for hemoperfusion. In general, the BSA immobilized 3D electrospun

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nanofiber sponge with rapid adsorption rate is an excellent adsorbent for hemoperfusion which can effectively reduce

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the hemoperfusion time while keep a high absorption capacity and have great potential used in some other biomedical applications.

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2. Materials and methods

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2.1. Materials

Polyacrylonitrile (PAN) was purchased from Sigma-Aldrich Co. Ltd.(China). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, ≥99%), glutaraldehyde (50 wt% in water), ethylene diamine (≥99.5%) and bovine serum albumin (BSA, ≥98%)

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were purchased from Innochem Technology Co. Ltd. (China). N,N-dimethylformamide (DMF, ≥99%), bilirubin, sodium hydroxide, sodium borohydride, potassium phosphate dibasic, potassium phosphate monobasic, sodium

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bicarbonate, sodium carbonate were all purchased from Aladdin Bio-Chem Technology Co. Ltd. (China). Enhanced BCA protein assay kit and cell counting kit-8 were from Beyotime Institute of Biotechnology (China). Total bilirubin (T-BIL) kit, prothrombin time assay Kit and activated partial thromboplastin time assay kit were from Jiancheng

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Biochemical Inc. (Nanjing, China). The plasma comes from volunteer (male, 26 years old). All buffers were prepared with ultra-pure Milli-Q water (resistance>18 MΩ cm-1). 2.2. Preparation of PAN nanofiber membranes PAN nanofiber membrane was prepared by electrospinning under optimum conditions. PAN electrospinning solution was prepared by dissolving PAN into DMF at a concentration of 12 wt% and stirring at room temperature 4

for 5 h until homogeneous. The solution was loaded into a 20 mL syringe equipped with a stainless-steel needle and discharged by a digitally controlled syringe pump, the flow rate and applied voltage were set at 2 mL/h and 15 kV. A aluminum foil covered plat（30 cm×30 cm）was used as collector and the distance between needle and collector was 15 cm. The experiment was carried out at 25℃ and 40% ± 2% relative humidity. All of the collected PAN nanofiber membranes were placed in vacuum overnight to remove the residual solvent.

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2.3. Preparation of the 3D electrospun nanofiber sponges 3D electrospun nanofiber sponges were prepared by an improved gas-foaming technique which depicted in Fig. 1a. The electrospun PAN nanofiber membranes were firstly immersed in sodium hydroxide solution (20 wt%) for 4

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h at 50℃(the membranes after oxidation are shown in Fig S1(a)), after thoroughly rinsed with ultra-pure water for 5 times，the membranes were protonated by immersed in hydrochloric acid solution (4 wt%) for 12 h at 37℃(the

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membranes after protonation are shown in Fig S1(b)). The modified (also pre-foamed) membranes were loose and had sufficient carboxyl groups. Then the membranes were expanded in sodium borohydride solution for 10min. At

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last, the scaffolds were thoroughly rinsed with ultra-pure water for 5 times and freeze-dried, then the 3D electrospun

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nanofiber sponges were obtained.

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2.4. Surface functionalization of nanofiber on the sponge

The obtained 3D electrospun nanofiber sponges were abundant with carboxyl groups, ethylene diamine was

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used to introduce amino groups and then BSA molecules were immobilized on the nanofiber surface with glutaraldehyde as coupling agent. The modification and immobilization processes are depicted in Fig. 1b. The

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carboxyl-containing sponge was weighed for 50mg and washed with 20 mL purified water for 3 times，and then with 0.2 M sodium phosphate buffer (20 mL; pH=5.8) for 3 times. After that, the sponge was immersed in EDC/NHS

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solution (20 mg/ml in 0.2 M sodium phosphate buffer; mole ratio 1:1; 20 mL; pH=5.8) for 4 h. Next, the sponge was immersed in ethylenediamine solution (0.5 M in 0.2 M sodium phosphate buffer; 30 mL; pH=5.8) for 4 h, after rinsed with water until the pH was close to neutral and freeze-dried, the amino groups modified sponge was obtained. To

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obtain the BSA immobilized scaffold, the amino groups modified sponge was washed with 0.1 M sodium phosphate buffer (20 mL; pH=7.2) for three times. And then immersed in glutaraldehyde solution (5% v/v in 0.1 M sodium phosphate buffer; pH=7.2; 30 mL) for 5 h. After that, it was immersed in solution of BSA (0.1M, 0.25M, 0.5M, 0.75M, 1M, 1.5M, 2M, 3M in 0.1 M sodium phosphate buffer; pH=7.2; 30 mL) for 24h at 4℃. Finally, the BSA immobilized sponge was immersed in freshly prepared NaBH4 solution (0.1 M in sodium carbonate buffer, pH=9.2; 5

30 mL) for 2 h to reduce Schiff's base formed between the protein and the aldehyde on the scaffold and freeze-dried after thoroughly rinsed with ultra-pure water for 5 times. The carboxyl groups abundant, amino groups modified and BSA immobilized 3D electrospun nanofiber sponges were named PAN-COOH, PAN-NH2 and PAN-BSA, respectively. 2.5. Characterization of 3D nanofiber sponge. 2.5.1. Expansion height

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To investigate the relationship between the expansion height and the concentration of sodium borohydride. The PAN nanofiber membrane was cut into square pieces with side length a=1 cm, after pre-foamed processes, the

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membrane pieces were immersed in various concentrations of sodium borohydride (0.01M, 0.02M, 0.05M, 0.1M, 0.2M) for foaming expansion for 10 min. After thoroughly washed with ultra-pure water, the scaffolds were freezedried. The foaming heights were measured by vernier caliper for three times at different positions, and took average

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value.

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2.5.2. Porosity and Density

𝑉−𝑉0 𝑉

* 100 %

(1)

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ε =

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The porosity of the nanofiber scaffolds was calculated according to the change of volume of nanofiber scaffolds before and after expansion. Porosity was calculated with the following equation:

where ε is porosity, V is the volume of nanofiber scaffold, V0=m0/ρ0 is the calculated volume of bulk PAN material, m0 is the mass of bulk PAN material, and ρ0 is the density of bulk PAN material.

m

(2)

a2·h

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ρ = m/v =

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The densities (ρ) of calculated by the following equation:

Here, m, v, a, and h stand for mass, volume, average side length and height of the scaffold, respectively.

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2.5.3. Morphology

The morphology of PAN nanofiber membrane, modified nanofiber membrane and 3D nanofiber sponge were examined using a digital camera (Canon EOS 1300D, Japan) and scanning electron microscope (SEM, HITACHI-

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8010, Japan). Prior to evaluation, the fibers were coated with gold. 2.5.4. ATR-FTIR tests Attenuated total reflectance Fourier transformation infrared (ATR-FTIR, Bruker EQUINOX 55 FTIR, Germany)

spectroscopy was utilized to detect the modification processes on the nanofiber surface with a wave number range of 4000–500 cm-1. 2.5.5. BSA immobilization amount 6

The amount of immobilized BSA on the nanofiber surface was calculated by BCA enhanced BCA protein assay kit (Beyotime, China). The absorbance was measured by microplate reader (Biochrom Anthos 2010, UK). 2.6. Adsorption isotherm. To make the adsorption isotherms for bilirubin, PAN-COOH, PAN-NH2 and PAN-BSA all weighted for two samples of 200 mg and they were respectively dispersed into 20 mL bilirubin aqueous solution and bilirubin loaded

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plasma with different bilirubin concentrations(5, 10, 15, 20, 30 and 40 mg·dL-1)，after adsorption for 4 h, the concentration of bilirubin in aqueous solution and plasma was determined by total bilirubin (T-BIL) kit and

Qe =

(Ci-Ce)V

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microplate reader. The equilibrium adsorption amounts Qe (mg·g-1) were calculated with the following equation: (3)

m

where Ci (mg·mL-1) and Ce (mg·mL-1) were the initial and equilibrium bilirubin concentration respectively. V (mL)

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was the volume of bilirubin aqueous solution or bilirubin loaded plasma and m (g) was the mass of the sample. 2.7. Adsorption kinetics

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To investigate the adsorption kinetics of relevant samples for bilirubin, PAN-COOH, PAN-NH2 and PAN-BSA

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all weighted for two samples of 500 mg and they were respectively dispersed into 50 mL bilirubin aqueous solution

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(30 mg/dL) and bilirubin loaded plasma (30 mg/dL), then oscillated for 20, 40, 60. 80, 100, 120 min at room temperature. At each set time point, the concentrations of bilirubin were determined by total bilirubin (T-BIL) kit and

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microplate reader and the bilirubin adsorption mass was calculated with equation (2). 2.8. Dynamic adsorption of bilirubin

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The dynamic adsorption was carried out using a peristaltic pump and a laboratory made perfusion device in Fig.7(a). The construction of the perfusion device is as follows: the perfusion column is the outer barrel of the

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syringe after remove the piston part. The nanofiber sponges are placed inside the column. The end of the column with large diameter is connected with the variable diameter joint. Then the other ends of the column and the variable diameter joint respectively connected to the silicone tube for connecting the peristaltic pump. 500mg of the PAN-

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COOH, PAN-NH2 and PAN-BSA samples were respectively placed in the column and 50ml of the bilirubin aqueous solution or bilirubin loaded plasma with bilirubin concentration of 30 mg/dL was circulatory flowed through the column at a rate of 1 mL·min-1. The bilirubin concentrations were determined after 0 min, 20 min, 40 min, 60 min, 80 min, 100 min, 120 min circulation，meanwhile, the total protein concentration of the plasma at each set time point was also determined by enhanced BCA protein assay kit to investigate the effect of samples on protein in plasma. 7

2.9. Coagulation times PT (prothrombin time) and APTT (active partial thromboplastin time) were determined by using Prothrombin time Assay Kit and Activated Partial Thromboplastin time Assay Kit and the operation steps were accurately carried out according to the instructions. The samples of PAN-COOH, PAN-NH2, PAN-BSA were firstly placed in a clean centrifuge tube, and the platelet poor plasma (PPP; obtained by centrifuging blood at 3000 rpm for 15 min) was used

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to incubate with the samples at 37℃ for 0.5h. 50 μL of the PPP was taken for PT and APTT tests each time. 2.10. In vitro cytocompatibility Assay

Mouse 3T3 fibroblasts cells (ATCC) were cultured in DMEM (Hyclone, USA) supplemented with 5% FBS.

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Cells were maintained in a 5% carbon dioxide incubator at 37 ℃. All the PAN-COOH, PAN-NH2, PAN-BSA samples were decontaminated under UV light for 2 h. For cell proliferation study, 1 X 104 3T3 cells were seeded on sponges and cultured for 48h, cell viability was measured using CCK-8.

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3.1. Expansion height and density of the 3D nanofiber sponge

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3. Results and discussion

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As the nanofiber membrane expanded on the third dimension, the expansion height which related to the concentration of sodium borohydride can be used to indicate the degree of expansion. The thickness of PAN nanofiber

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membrane and pre-foaming nanofiber membrane, the expansion height of the nanofiber sponge expanded in 0.01M,

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0.02M, 0.05M, 0.1M, 0.2M NaBH4 solution are shown in Fig. 2(a). The thickness of electrospun PAN nanofiber membrane and pre-foamed nanofiber membrane were respectively 0.21mm and 0.96mm. Due to the ammonia gas produced on the surface of nanofiber during the process of modification, the nanofibers separated with each other

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and the membrane had a certain extent of expansion. This helped to the subsequent foaming expansion in sodium borohydride solution. The treated nanofiber membrane was easier to expand and the expansion degree had also

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become more controllable. A 10 min process time was enough to obtain the 3D nanofiber sponge. The expansion heights were related to the concentration of NaBH4 solution. In 0.01M, 0.02M, 0.05M, 0.1M, 0.2M NaBH4 solution, the expansion heights were respectively 2.16mm, 3.95mm, 11.88mm, 19.21mm, 29.76mm and the density of the 3D

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nanofiber sponge were respectively 139.44 mg/cm2, 76.48 mg/cm2, 25.40 mg/cm2, 15.67 mg/cm2, 10.09 mg/cm2. The expansion height increased with the concentration of NaBH4 solution while the density decreased with it. The porosities of the corresponding nanofiber scaffolds are shown in Fig. 2(b), the porosity increased with the increased expansion height. 3.2. Morphological analysis 8

SEM image and digital photograph of electrospun PAN nanofiber membrane are shown in Fig. 1(a). It can be found that the PAN nanofibers were randomly oriented with the diameters ranged from 100 to 400 nm. The modified nanofiber membrane became wrinkled and loose and the nanofibers became curled after pre-foaming (modification) process which can be seen from Fig. 1(b). The cross section of 3D nanofiber sponge in low and high magnifications are displayed in Fig.1 (c) (d). Layered structures can be found on the sponge and slender pores were formed by the interlaced layers. The thickness of the layer is about several fiber diameters, and the fibers still keep densely packed

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on the layer while sparse fibers cross the gaps. The thickness of the layer is thinner and the stratification is more uniform commpared to the previous literatures[26] ,which is related to the pre-foaming process.

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3.3 ATR-FTIR

The modification from -C≡N to -COOH on PAN nanofiber and the bonding of ethylenediamine with –COOH were confirmed by FTIR analysis (Fig. 4a). The three curves in the graph are correspond to PAN nanofiber (PAN), -

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COOH modified nanofiber (PAN-COOH) and –NH2 modified nanofiber (PAN-NH2) respectively. The absorption

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peak at 2242 cm-1 corresponded to the stretching vibration of -C≡N，and the peak at 2935 cm-1 belonged to the -CH2-

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stretching vibration on PAN spectrum. This two peaks could be also found in PAN-COOH and PAN-NH2 spectrums

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which indicates that the -C≡N groups on the nanofiber surface were partially modified. On PAN-COOH spectrum, the absorption band at 3372 cm-1 and absorption peak at 1730 cm-1 were respectively attributed to –OH and –C=O

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stretching vibration of -COOH. Confirming that the -C≡N had been successfully modified to –COOH. The wide absorption band at 2650-3670 cm-1 on PAN-NH2 spectrum was arose from the -N-H stretching vibration of -CO-NH-

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and –NH2. The absorption at 1645 cm-1, 1567 cm-1 and 1452 cm-1 correspond respectively to the –C=O stretching vibration, -N-H bending vibration and -C-N stretching vibration of the -CO-NH- while the absorption at 1328 cm-1

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and 1064 cm-1 was assigned to -C-N stretching vibration and -N-H bending vibration of –NH2. These results confirmed the modifying processes depicted in Fig.1. 3.4 BSA immobilization

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For BSA immobilization, glutaraldehyde was used as a cross-linker to connect the amino on the sponge and amino of BSA. The effect of BSA concentration was investigated over the range of 0.1–3.0 mg/mL and the amount of immobilized BSA increased with the increase of BSA concentration until 1.5mg/ml then remained roughly constant (Fig. 4(b)), the final equilibrium value was 44.42 mg/g. 3.5 Adsorption isotherm The adsorption isotherms of bilirubin on PAN-COOH, PAN-NH2 and PAN-BSA in aqueous solution and plasma 9

are shown in Fig.5 (a) (d) and the equilibrium adsorption capacity at different initial concentrations are listed on supplementary table s1. The equilibrium adsorption capacity of all samples increased with the bilirubin initial

concentration (basically linear) in aqueous solution. The adsorption capacity of PAN-BSA was the largest, followed by PAN-NH2 and PAN-COOH. In plasma, the equilibrium adsorption capacity of PAN-COOH is small, only 4.3499 mg/g even at the concentration of 40 mg/ml. For PAN-NH2, the adsorption amount reached equilibrium when the initial concentration reached 20 mg/dL. While, the performance of PAN-BSA in plasma is basically the same with in

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aqueous solution except the amount of adsorption was reduced a certain extent. In general, at each initial concentration, the equilibrium adsorption capacity of all samples in the plasma was lower than that in the aqueous

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solution. This may be related to the tight binding of bilirubin to serum albumin in the plasma.

In order to understand the adsorption mechanism better, the experimental data were simulated by Langmuir, Freundlich isotherm equations, respectively. The Langmuir isotherm model was suitable for monolayer adsorption

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onto a surface with a finite number of identical sites, and the Freundlich isotherm model was used for heterogeneous

qe

=

1 qmaxKL

Freundlich isotherm: lnqe =

+

lnKF +

Ce qmax lnCe n

(4) (5)

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Ce

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Langmuir isotherm：

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systems. The two isotherm models were mathematically described as follows:

where qmax was the maximum adsorption capacity, qe was the bilirubin adsorption amount at different initial

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concentrations, Ce was the equilibrium bilirubin concentration, KL was the Langmuir isotherm constant, KF was the Freundlich constant depicting adsorption capacity, and 1/n was the heterogeneity factor indicating adsorption

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intensity.

The Langmuir adsorption isotherm and the Freundlich adsorption isotherm for the relevant samples are shown

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in Fig. 5 b, c (in aqueous solution) and Fig. 5 e, f (in plasma) respectively, and the calculated isotherm parameters are listed on supplementary table s2 and table s3. It could be seen that, in aqueous solution, the correlation coefficients of the Langmuir adsorption isotherm for PAN-COOH, PAN-NH2 and PAN-BSA were 0.6004, 0.5839 and 0.5553

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respectively, while the correlation coefficients of the Freundlich adsorption isotherm for PAN-COOH, PAN-NH2 and PAN-BSA were 0.9238, 0.9427 and 0.9409 respectively. The results indicated that the adsorption process of bilirubin matches the Freundlich model better than the Langmuir model in the studied concentration range. In plasma, the correlation coefficients of the Langmuir adsorption isotherm for PAN-COOH, PAN-NH2 and PAN-BSA were 0.2910, 0.8488 and 0.3494, meanwhile 0.9611, 0.8510, 0.9543 of the Freundlich adsorption isotherm. PAN-COOH and PAN10

BSA match the Freundlich model well while PAN-NH2 matches the Langmuir model better. The Bilirubin is unstable in aqueous solution and easy to be adsorbed on the surface of the nanofiber samples, the adsorption had been less affected by the surface groups. In plasma, the adsorption of bilirubin is relatively difficult for PAN-COOH. The amino group on the PAN-NH2 had an adhesion to bilirubin and basically monolayer adsorption. PAN-BSA still had a good adsorption effect in plasma, a 1/n value of 0.8480 indicating that it is a favorable absorbent for bilirubin. 3.6. Adsorption kinetics

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The kinetics of adsorption of bilirubin on PAN-COOH, PAN-NH2 and PAN-BSA in aqueous solution and plasma at different adsorption times from 20 to 120 min were examined. The initial concentrations of bilirubin were both set

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as 30 mg/dL. Bilirubin concentration at different adsorption time in aqueous solution and plasma were respectively shown in Fig.6 (a) (d). In aqueous solution, all the samples had a rapid adsorption rate, the adsorption reached equilibrium at 60 min for the PAN-BSA while the PAN-COOH and PAN-NH2 had an equilibrium time about 40 min.

In plasma, PAN-COOH and PAN-NH2 had a weak adsorption to bilirubin (2.6883mg·g-1 and 5.9909 mg·g-1), yet

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The final adsorption capacities of PAN-COOH, PAN-NH2 and PAN-BSA were 18.3035, 22.6845 and 27.1003 mg·g-

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the PAN-BSA had a relatively good result (18.5323 mg·g-1) but lower than in aqueous solution. In addition, adsorption kinetic data collected were analyzed with pseudo-first order kinetic and pseudo-second order kinetic models in order

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to clarify the adsorption kinetics of the bilirubin onto the samples surfaces. The equations were taken in their linear

Pseudo-first-order kinetic model:

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forms as follow :

ln(qe - qt) = lnqe - k1t

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Pseudo-second-order kinetic model:

t

qt

=

1

k2qe

2 +

t qe

(6) (7)

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where qt (mg/g) was the adsorption amount at a given contact time t (min), qe was the calculated saturation capacity, k1 and k2 were the relevant rate constant of the two kinetics models, respectively. The fitted curves based on these two kinetics models in aqueous solution and plasma are respectively displayed

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in Fig.6 (b) (c) and Fig.6 (e) (f), and the corresponding kinetic parameters were calculated and listed on supplementary table s4 and table s5. In aqueous solution, the kinetic data of PAN-COOH and PAN-NH2 showed a better fit to the pseudo-second-order kinetic model with the R2 values of 0.9898 and 0.9979 respectively, and PAN-BSA fit well to both Pseudo-first-order kinetic model (R2=0.9847) and pseudo-second order kinetic model (R2=0.9996) simultaneously. That may indicate that the adsorption processes on PAN-COOH and PAN-NH2 in aqueous solution were chemisorption and the adsorption process on PAN-BSA was dominated by both physisorption and 11

chemisorption. In plasma, owing to the bad adsorption effect, PAN-COOH can't fit either of these two models. While, PAN-NH2 (R2=0.9888) and PAN-BSA(R2=0.9705) had good fit results with pseudo-second order kinetic model. The adsorption processes were mainly chemisorption. Combination of the above results, PAN-BSA had the best adsorption effect on bilirubin both in aqueous solution and plasma. According to the correlation coefficients of kinetic models, chemical adsorption is the dominant factor for all these samples. 3.7. Dynamic adsorption

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To simulate clinical hemoperfusion equipment for the treatment of hyperbilirubinemia, a laboratory-made perfusion column (Fig. 7a) connected with peristaltic pump was used to perform dynamic experiments. Bilirubin

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aqueous solution and bilirubin loaded plasma circulatory flowed through the perfusion columns respectively. The results of dynamic adsorption of relevant samples are shown in Fig. 7 b, c. When the flowing liquid was bilirubin aqueous solution, the concentration of the bilirubin decreased rapidly during the first 60 min, and then kept stable.

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The final concentration of bilirubin in aqueous solution after dynamic adsorption with PAN-COOH, PAN-NH2 and

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PAN-BSA were 12.9004 mg/dL, 8.7953 mg/dL and 5.1277 mg/dL respectively. For PAN-COOH and PAN-NH2, the

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dynamic adsorption results in plasma were bad, the bilirubin concentrations only declined little. While, PAN-BSA had a good performance, bilirubin concentration decreased from 30 mg/dL to about 11.9085 mg/dL after 120 minutes

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circulation. It indicated that the PAN-BSA had great potential to be used as the adsorption materials for

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hemoperfusion treatment on hyperbilirubinemia. Total protein contents in plasma were determined during the dynamic absorption process to investigate the effect of samples on the protein in plasma (Fig.7 d). PAN-COOH, PAN-NH2 and PAN-BSA were all had certain adsorption to the proteins in the plasma, thanks to strong hydrophilicity

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of these three samples, the adsorption amount was small and in acceptable range. 3.8 Cell viability and Clotting time assays

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To test the cytotoxicity of relevant samples, the influence of PAN-COOH, PAN-NH2 and PAN-BSA over mouse fibroblast cells (3T3 line) viability was determined by CCK-8 assay after culturing for 1 d and 2 d. As shown in Fig. 8a, no significant differences could be observed of the cell viability for all the samples after 2 days cell culture in

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comparison with the blank control. Cells could grow and proliferate well on the samples, and the samples had no cytotoxicity. Activated partial thromboplastin time (APTT) and prothrombin time (PT) are the global screening procedures which are usually used to evaluate coagulation abnormalities in the intrinsic pathway and extrinsic pathway, respectively. These two indexes can also be used to detect functional deficiencies in Factor II, III, V, VIII, X, or 12

fibrinogen.[32] When an anticoagulative material contacts with blood, it may combine or react with the coagulation factors, and APTT or PT could be prolonged. Fig. 8 b shows the APTTs and PTs of PAN-COOH, PAN-NH2 and PANBSA. From this figure, it can be found that APTTs for PAN-COOH and PAN-NH2 were prolonged while no change for PAN-BSA. The increment of APTTs for PAN–COOH and PAN-NH2 may be attributed to the combination or reaction between the functional groups (–COOH and –NH2) and the coagulation factors mentioned above. There was no obvious change in PTs compared with the blank control group for all three samples. The results indicated that

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PAN-BSA had a good blood compatibility while no effect on the blood coagulation factors. There was certain anticoagulant property for PAN-COOH and PAN-NH2 samples.

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4. conclusions

In conclusion, we have developed a 3D nanofiber sponge with special layered structure and high porosity used for bilirubin removal in hemoperfusion. The 3D nanofiber sponge was fabricated by combination of electrospinning

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and improved gas-foaming techniques. The results showed that the BSA-immobilized 3D nanofiber sponge had high

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adsorption efficiency and rapid adsorption rate. It also showed a good performance in dynamic adsorption and a good

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blood compatibility. In general, the as-prepared BSA-immobilized 3D nanofiber sponge with rapid adsorption rate is an excellent adsorbent for hemoperfusion which can effectively reduce the hemoperfusion time while keep a high

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absorption capacity. This method could be extended to the preparation of more other materials and showed great

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Conflicts of interest

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potential for more widely biomedical applications.

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There are no conflicts of interest to declare

Acknowledgments

The authors would like to thank Beijing Natural Science Foundation (2172039), the National S&T Major Project of

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China (SQ2018ZX100301), NSFC (51373023), the Open Research Fund Program of Key Laboratory of Cosmetic (Beijing Technology and Business University), China National Light Industry (KLC-2017-YB5), and the Fundamental Research Funds for the Central Universities

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Fig. 1 (a) Schematic showing the 3D electrospun nanofiber sponge fabrication steps; (b) modification and immobilizing processes on the nanofiber surface of the sponge.

Fig. 2 (a) Expansion height of nanofiber membrane in NaBH4 solution with different concentrations and (b) . corresponding porosity to the materials

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bar of the inset image is 4 µm).

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Fig. 3 SEM images showing the representative morphological structures of (a) PAN nanofiber membrane; (b) modified nanofiber membrane; 3D nanofiber sponge cross section in (c) low and (d) high magnifications (scale

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Fig. 4 (a) FTIR spectrums of PAN nanofiber, carboxyl modified nanofiber and amino modified nanofiber; (b) BSA immobilization amount on 3D nanofiber sponge in BSA solution with different concentrations.

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Fig. 5 (a)Bilirubin adsorption performance in aqueous solution, the fitting (b) Langmuir isotherms and (c) Freundlich isotherms of bilirubin on PAN-COOH, PAN-NH2, PAN-BSA samples. (d)Bilirubin adsorption performance in plasma, the fitting (e) Langmuir isotherms and (f) Freundlich isotherms of bilirubin on PANCOOH, PAN-NH2, PAN-BSA samples.

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Fig.6 (a) Adsorption kinetics, (b) the pseudo-first-order kinetic model and the (c) pseudo-second-order kinetic model of the PAN-COOH, PAN-NH2, PAN-BSA samples for bilirubin adsorption in aqueous solution. (d) Adsorption kinetics, (e) the pseudo-first-order kinetic model and the (f) pseudo-second-order kinetic model of the PAN-COOH, PAN-NH2, PAN-BSA samples for bilirubin adsorption in plasma.

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Fig. 7 (a) digital photograph of laboratory-made perfusion column; dynamic adsorption performance of relevant samples (b) in aqueous solution and (c) in plasma; (d) total protein contents in plasma during the dynamic adsorption.

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Fig. 8 (a) cell viability and (b) coagulation time of PAN-COOH, PAN-NH2, PAN-BSA samples.

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