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Journal of Membrane Science 405– 406 (2012) 104– 112

Contents lists available at SciVerse ScienceDirect

Journal of Membrane Science

j ourna l ho me pag e: www.elsev ier .com/ locate /memsci

xtracorporeal endotoxin removal by novel l-serine grafted PVDF membraneodules

o Zhanga, Lin Zhanga, Li-Hua Chengb,∗, Kun Xua, Qiu-Ping Xuc, Huan-Lin Chena, Juin-Yih Laid,uo-Lun Tungd

Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, PR ChinaDepartment of Environmental Engineering, Zhejiang University, Hangzhou 310058, PR ChinaCritical Care Department, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, PR ChinaR&D Center for Membrane Technology and Department of Chemical Engineering, Chung Yuan University, Chungli, Taiwan

r t i c l e i n f o

rticle history:eceived 6 January 2012eceived in revised form 22 February 2012ccepted 24 February 2012vailable online 6 March 2012

eywords:ndotoxin

a b s t r a c t

In this work, two sizes of affinity membrane modules for extracorporeal elimination of endotoxin fromblood were prepared by grafting l-serine (Ser) ligand onto PVDF hollow fiber membrane. The chemicalmodification of membrane surface was verified by X-ray photoelectron spectroscopy (XPS) analysis. Themodified fiber’s ability to adsorb endotoxins was examined in aqueous solutions with varied pH valueand ionic strength, while the protein adsorption of this fiber was investigated in BSA solution. The small-sized module with 159.4 cm2 effective area was used to remove endotoxin from sepsis patients’ plasmain vitro. As results, when the original concentration was 0.42 EU/ml, the endotoxin adsorption capability

2

VDF-Serine (Ser)ollow fiberemoperfusion

was 0.058 EU/cm and the removal efficiency was almost 100% for 15 ml, 93.5% and 48.3% for 20 ml and40 ml samples, respectively. The recovery ratios of total protein and albumin were achieved up to 85.1%and 92.4% correspondingly. The pig model was finally established to evaluate the endotoxin removalefficacy of the scaled up module in direct hemoperfusion. The endotoxin and the inflammation mediatorlevels decreased after hemoperfusion treatment, indicating the promising potential of this novel adsorberfor blood purification of endotoxin.

. Introduction

Endotoxin, the lipopolysaccharide (LPS) component of the outerell wall of Gram-negative bacteria, produces serious biologicalffects on human beings and other mammals when it enters into thelood system, even at extremely low concentration. It is considereds a key factor in the pathogenesis of sepsis or severe sepsis [1]. Theate of severe sepsis hospitalization increases continuously duringast decades and the general mortality is extremely high both in theSA and Europe in the intensive care units (ICUs) due to the lack offfective therapies [2,3]. Great efforts have been made on the in vivoreatment, but the clinical outcomes are not satisfying becausef poor immunogenicity and inaccessibility of antibodies as wells possible tissue damage induced by drugs and disappointinghase III trial results [4,5]. Hence the direct blood purifica-ion method begins to attract attention as a potential promising

ay.

During the past two decades, hemoperfusion had shown its sig-ificance in sepsis therapy as a kind of continuous extracorporeal

∗ Corresponding author. Tel.: +86 571 88982025; fax: +86 571 88982025.E-mail address: chenglihua@zju.edu.cn (L.-H. Cheng).

376-7388/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.memsci.2012.02.057

© 2012 Elsevier B.V. All rights reserved.

treatment approach, where the circulating endotoxins and immunemediators in blood could be removed by selective adsorption. Sev-eral extracorporeal adsorbers, such as Matisse® adsorber (FreseniusHemocare Adsorber Technology GmbH, Germany) [6,7], H.E.L.Psystem (B. Braun, Germany) [8], Toraymyxin (Toray IndustriesInc., Japan) [9] and CTR column [10], are now under investiga-tion in clinical applications and have been proved to possess thecapability of removing endotoxins from severe sepsis patients’plasma. For instance, over 30,000 sepsis and septic shock patientshave been treated by Toraymyxin cartridge in Japan, in whichpolymyxin B was immobilized onto polystyrene fabric. The car-tridge is considered to be able to remove circulating endotoxinand improve hemodynamic dysfunction [9]. Despite the extra-corporeal adsorption approach can be an appropriate choice forsepsis therapy, polymyxin B is highly expensive and its nephro-toxicity and neurotoxicity are also crucial issues. Additionally, themain limiting factor is its removal efficiency, as the general reduc-tion of plasma endotoxin level of those reported adsorbers is only30–40% [7–9], indicating the necessity to develop alternative prod-

ucts which are characterized with low cost, high safety and highefficiency.

To remove effectively the endotoxin from blood, the lig-ands immobilized on matrices are expected to bind endotoxin

rane S

scpa[rb(plhclkat(sbclStSsbiur

adgaDfifalttma

eaiadpiwsfit

PlwaAcuari

troscopy (XPS) (AXIS-UlTRA DLD Kratos Co., England). The amountof amino acid coupled on the PVDF-Ser membrane was deter-

M. Zhang et al. / Journal of Memb

electively with a low non-specific adsorption. Endotoxin ishemically composed of a polar polysaccharide chain and a non-olar partially phosphorylated domain called lipid A, which ismphiphilic and is the most conservative part of endotoxin11]. Most adsorbents are designed to interact with lipid Aegion through the electrostatic and hydrophobic interactionsetween the ligands and endotoxin, like diethylaminoethaneDEAE) [12], polymyxin B (PMB) [13], histidine [14], lysine [15],oly(ethyleneimine) (PEI) [16], poly(l-lysine) (PLL) [17], dimethy-

amine [18] and chitosan [19]. However, the electrostatic andydrophobic interactions exist simultaneously between thoseationic ligands and acidic proteins, which results in high proteinoss. Therefore, the ligands which can adsorb endotoxin by otherinds of interactions are desirable. Recently, Wei et al. [20] prepared

series of adsorbents with different amino acid ligands for endo-oxin removal, and found that the removal efficiency of l-serineSer) was up to 78% in rabbit’s serum. The results from the computerimulation clearly showed that a firm cage structure was formedetween endotoxin phosphoric residue and Ser adsorbent via threeouples of hydrogen bonds with the assist of hydroxyl group on theateral chain of Ser ligand, NH group on the linkage site betweener and space arm, and hydroxyl group on space arm, indicatinghe importance of hydrogen bond and steric effect in the process ofer ligand binding to endotoxin. Besides, Ser is much cheaper andafer than PMB. Thus, Ser might be suitable for application in thelood system. Nevertheless, the conventional agarose column used

n Wei’s work limits its practical application due to low sample vol-me. Therefore, further research on the adsorption module is stillequired to improve its configuration.

Alternatively, hollow fiber affinity membrane module, withdvantages of high flow rate, large throughput, low axial-pressurerop and easy scale-up [21], can also selectively adsorb the tar-et compound when specific ligands are grafted. It has beenpplied in the depyrogenation operation of protein solutions andNA samples [22,23]. Some surface-modified hemodialysis hollowber membranes are also reported to have ability to remove LPS

rom blood [24,25]. Compared with traditional adsorbers whichre designed as columns packed with functionalized beads, hol-ow fiber affinity membrane can reduce the total resistance ofhe module to obtain a stable blood flow, which is beneficialo the hemoperfusion process. Therefore, hollow fiber affinity

embrane modules are potential for the practical hemoperfusionpplication.

Poly(vinylidene fluoride) (PVDF), with good mechanical prop-rty and extraordinary chemical stability, has attracted increasingttention as a promising biomaterial due to its excellent biocompat-bility [26]. On the other hand, the application of PVDF membranes the blood-contact material is limited by its hydrophobic natureue to the simultaneous non-specific adsorption of proteins. Ourrevious work had provided an effective modification method to

ncrease the hydrophilicity of PVDF [27]. The PVDF hollow fibersith hydroxyethyl cellulose (HEC) coating have shown a low non-

pecific adsorption in human plasma. Therefore, the PVDF hollowber membranes are grafted with l-Ser after hydrophilic modifica-ion and further studied for the endotoxin removal from blood.

In this work, the novel adsorbents were prepared by usingVDF hollow fibers as supported material and l-Ser as theigand. The static specific adsorption behavior of the adsorbent

as then studied in an aqueous solution, and the non-specificdsorption was investigated using BSA as the objective protein.fterwards, the endotoxin clearance efficiency and the hemo-ompatibility of the affinity membrane module were evaluated

sing the sepsis patients’ plasma where the endotoxin level wasround 1 EU/ml. Finally the hemoperfusion experiment was car-ied out using pig model and the therapy results were furthernvestigated.

cience 405– 406 (2012) 104– 112 105

2. Materials and methods

2.1. Materials

The PVDF hollow fiber membranes used in this study wereprepared in our laboratory. The inside and outside diameters ofthe fibers were 0.8 and 1.1 mm respectively, with the averagepore size of 0.18 �m, the porosity of 85%, and the water flux(0.1 MPa) of 69.71 ml/cm2 min. HEC was purchased from Fluka(Buchs, Switzerland). 1, 6-Hexanediamine (HDA), as the space arm,was from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).While, amino acid l-serine was bought from Shanghai Kangda Co.(Shanghai, China). Deionized water was used throughout the exper-iments.

Bovine serum albumin (BSA, Mw 67,000, pI = 4.7) was pur-chased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,China). Escherichia coli 0111: B4, L2630 (Sigma–Aldrich, USA) wasused as the endotoxin standard samples. Quantitative chromogenictachypleus amebocyte lysate (TAL) for endotoxin detection (theminimum detection limit is 0.01 EU/ml) and endotoxin-free waterwere purchased from Xiamen Horseshoe Crab Reagent Manufac-tory Co., Ltd. (Xiamen, China). Human plasma was supplied byvolunteer sepsis patients in Sir Run Run Shaw Hospital, School ofMedicine, Zhejiang University.

Male domestic pigs weighing 25–30 kg were used as the animalexperimental body. The animal preparation was referred to the pre-vious study [28]. ELISA kits for tumor necrosis factor-� (TNF-�) andinterleukin-6 (IL-6) testing were purchased from Bluegene BiotechCO., Ltd. (Shanghai, China).

All other chemical and biological reagents were of analyticalgrade from local chemical reagent companies. All the glass appa-ratuses were heated for 4 h at 400 ◦C and plastic apparatuses wereimmersed in 30% H2O2 to keep pyrogen-free conditions throughoutthe endotoxin adsorption experiments.

2.2. Affinity membrane preparation and characterization

PVDF membrane was chemically modified as shown in Fig. 1[27]. The PVDF hollow fibers were treated with 4% (w/v) KMnO4and 3 mol/L KOH at 80 ◦C for 30 min, subsequently were immersedin 5% (v/v) H2SO4 and 5% (w/v) NaHSO3 at room temperature tillthey turned white (step 1). The hydroxylated membranes werethen shaken in 1.5% (w/v) HEC (pH 6–7, adjusted by NaOH) at90 ◦C for 15 min to increase the reactive hydroxy groups of thesurface. After dried, the membranes were washed with 0.5 mol/LNaHCO3 at 90 ◦C (step 2). Thereafter, the HEC-bound membraneswere treated with 4:1 (v/v) mixture of 1.0 mol/l NaOH and chloroe-poxy propane at 60 ◦C for 1 h (step 3). Then the membranes wereshaken in 5% (w/v) 1,6-hexanediamine (HDA) at 60 ◦C for 2 h tofix spacer arms (step 4). After being treated with chloroepoxypropane again (step 5), the resulting membranes were shaken in3.0 mg/ml l-serine (Ser) at 45 ◦C for 24 h (step 6), which was pre-pared in 0.2 mol/L sodium phosphate buffer (pH 7.2). Finally, themembranes were washed with 0.2 mol/L sodium phosphate bufferand deionzied water, followed by drying process and stored for lateruse.

The surface properties of PVDF membrane and PVDF-Seraffinity membrane were analyzed by X-ray photoelectron spec-

mined by measuring the initial and final concentrations of theserine solution in step 6 using the ninhydrin method [29].The amount of Serine was calculated to be 4.42 �mol/cm2

membrane.

106 M. Zhang et al. / Journal of Membrane Science 405– 406 (2012) 104– 112

ion of

2

aewmw

E

wis

2

ici1ocBU

2

e

Fig. 1. Scheme of the preparat

.3. Static endotoxin adsorption experiment

Endotoxin content was quantitatively assayed by the tachypleusmebocyte lysate (TAL) test, measuring by S-54 UV–vis spectrom-ter (Shanghai Lengguang Tech. Co., Ltd.) at 545 nm. All samplesere measured in duplicate. The endotoxin removal ability of theembrane was evaluated by endotoxin adsorption capacity (EAC),hich was defined as:

AC = mo − mf

S(1)

here mo was the amount of endotoxin (in endotoxin unit, EU)n original solution, mf was the amount of endotoxin (EU) in finalolution, and S (cm2) was the surface area of the affinity membrane.

.3.1. Pre-test adsorption experimentThe experiments for endotoxin and protein adsorption capac-

ties of PVDF membrane and PVDF-Ser affinity membrane werearried out in BSA solutions. The membranes of 10 cm2 were putnto 10 ml BSA solutions (1 mg/ml, pH 6.5, endotoxin concentration.5–1.8 EU/ml) and shaken for 2 h at room temperature. The amountf adsorbed endotoxin and BSA was calculated by measuring theoncentrations of initial and final solutions. The concentration ofSA was determined by the absorbance at 278 nm with the S-54V–vis spectrometer.

.3.2. Effects of pH value and ionic strengthThe effects of pH and ionic strengths on the adsorption of

ndotoxin were measured by a batchwise mode. A series of

PVDF-Ser affinity membrane.

buffers were prepared by 0.25 M sodium acetate (pH 4, 5), 0.25 Msodium phosphate (pH 6–8), and 0.25 M Tris–HCl (pH 9, 10).Sodium chloride solutions were prepared with seven differentionic strengths (i.e. 0.05 mol/L, 0.10 mol/L, 0.15 mol/L, 0.20 mol/L,0.25 mol/L, 0.30 mol/L and 0.50 mol/L). The PVDF-Ser affinity mem-branes were put into various solutions and shaken for 2 h at roomtemperature. The initial endotoxin concentration and final endo-toxin concentration were measured and adsorption curves wereall averaged in triplicate.

2.3.3. Determination of adsorption isothermNormally the median endotoxin levels in patients with sepsis

were 3 EU/ml [30], so we did the endotoxin adsorption experimentsfor determination of its isotherm in the range from 0.5 EU/ml to3.0 EU/ml. The LPS solutions with endotoxin concentration rang-ing from 0.5 EU/ml to 3.0 EU/ml were prepared with pyrogen-freewater as the solvent. PVDF-Ser affinity membrane was cut into5 mm long and then decontaminated before the experiment. Mem-branes of 2 cm2 were put into 5 ml LPS solutions and shaken at roomtemperature for 2 h to investigate the effect of initial concentra-tion of LPS solutions on endotoxin adsorption behavior. The amountof adsorbed endotoxin was calculated to construct the adsorptionisotherm.

2.4. Endotoxin removal from

human plasma

The endotoxin adsorption capacity (EAC) of PVDF-Ser mem-brane in human plasma was evaluated in a lab-made module

M. Zhang et al. / Journal of Membrane Science 405– 406 (2012) 104– 112 107

(oachutthahora

niat

2

cPa6

(tuTt

1eOaToacece

800600400200

N (0.47% )

F (0.8% )

O (4.95% )C (51.12% )

C (71.86% )

N (2.02% )

F (43.47% )

O (20.10% )

PVDF membrane

Affinity membrane

The prepared Ser affinity membranes are expected to bind endo-

Fig. 2. Scheme of hollow fiber module for dynamic adsorption experiment.

Fig. 2). The glass-made module was packed with 16 hollow fibers,f which the effective length was 16.7 cm and the total effectiverea was 159.4 cm2. The plasma flowed along the direction as indi-ated by the red arrow in Fig. 2. According to this figure, 40 mleparinized plasma sample was pumped into the shell side of mod-le from the bottom inlet at a flow rate of 1.0 ml/min, running alonghe outside surface of the fibers. When the plasma moved up tohe top of the module, it recycled back into the lumen side of theollow fibers. The final outflow was collected every 3 min for TALssay to get the breakthrough curve. The EAC of the membrane inuman plasma was calculated by comparing the endotoxin levelf the original and the filtrate sample. Considering the difficulty ofegeneration and decontamination after plasma flowing through,ll the modules are only used once.

The bio-chemical tests were carried out on both sample (origi-al and filtrate), by using some basic components in human plasma

ncluding aminotransferase, total protein, albumin, bilirubin, cre-tinine, glucose and electrolyte as the indexes to learn the effect ofhe adsorption process on the plasma.

.5. Hemoperfusion experiment using pig model

The animal experiment was conducted on endotoxin-hallenged pig models to evaluate the efficacy and safety ofVDF-Ser adsorbers. The affinity membrane modules used innimal experiment were scaled up to reach an effective area of37.8 cm2.

Two experimental pigs were infused with endotoxin60 �g/kg/h) for 2 h. After endotoxin challenged, one was consecu-ively treated with direct hemoperfusion using PVDF-Ser adsorbernder anticoagulant infusion (maintained at 40 IU/kg/h) for 3 h.he blood flow rate was 25 ml/min. The other pig without anyreatment was designed as the control group.

The concentration of plasma endotoxin was measured every h for both experimental group and control group, while the lev-ls of TNF-� and IL-6 were also detected during the experiment.f all the cytokine proteins, TNF-� and IL-6 have received muchttention for their pathophysiological roles in infection and trauma.heir levels indicate the host defense and the triggering of a seriesf reactions involving multiple organs, and the excessive releaseslways imply severe local or systemic immune response, whichulminates in the “sepsis syndrome” [31]. Thus they are consid-

red to be promising candidate indicators for severe sepsis. Thehange of TNF-� and IL-6 may help to evaluate the hemoperfusionffect.

Binding Energy (ev)

Fig. 3. Analysis of surface composition by XPS.

3. Results and discussion

3.1. Membrane surface analysis

As schematically described in Fig. 1, PVDF hollow fibers werefirstly modified to be hydrophilic and then treated with chloroe-poxy propane and HDA, finally immobilized with l-Ser. To verify themodification effect, XPS survey scans were performed to analyzethe chemical structure changes of the membrane surfaces.

Fig. 3 demonstrates that, the Ser-affinity membrane shows obvi-ous changes in four main peaks of F, O, N and C compared with theoriginal PVDF. The fluorine content decreased from 43.47% to 0.8%,while the carbon content increased from 51.12% to 71.86%, the oxy-gen content increased from 4.95% to 20.10%, and nitrogen contentincreased from 0.47% to 2.02%. The decrease of F and increase of Oand C are due to the hydrophilic modification of the surface espe-cially the coating of HEC. The increase of N is mainly attributed tothe graft of HDA spacer arms and the immobilization of Ser lig-ands. Meanwhile, the slight O (4.95%) and N (0.47%) contents of theoriginal membrane are probably caused by solvent residues andpolymer contaminants.

As shown in Table 1, the C1s main peaks of the PVDF mem-brane and affinity membrane, positioned at 287.063 eV, 291.021 eV,284.800 eV, 289.017 eV, and 285.764 eV are assigned to the car-bons C* of units C*H2 CF2 , C*F2 , C*H2 CH(OH) , C*OOHand C*H2 NH , respectively. The relative areas of the differ-ent peaks represent the relative contents of different elements(Table 1). Compared with the PVDF membrane, the contents of C*in C*H2 CF2 and C*F2 of affinity membrane decreased from46.9% to 39.7%, and 31% to 2.4%, respectively, while the content of

C*H2 CH(OH) increased from 8.5% to 44.2%. These changes indi-cate the existence of HEC and HDA on the membrane surface, andthe successful hydrophilic modification and space arm graft. Theincrease of C*H2 NH of affinity membrane (5.7%) is due to thebonding of spacer arms and ligands. The occurrence of C*OOH upto 8.0% shows that the successful ligands immobilization onto thePVDF membrane.

3.2. Endotoxin adsorption experiments

3.2.1. Preliminary tests on endotoxin and BSA adsorption

toxin selectively whereas to exhibit a low non-specific adsorption.Therefore, pre-test adsorption experiments were carried out toevaluate the specific and non-specific adsorption capacities of both

108 M. Zhang et al. / Journal of Membrane Science 405– 406 (2012) 104– 112

Table 1Analysis of C1s by XPS.

Sample C*H2 CF2 C*F2 C*H2 CH(OH) C*OOH C*H2 NH

Eb (eV) w% Eb (eV) w% Eb (eV) w% Eb (ev) w% Eb (ev) w%

otbiovw9ittaotdt

otficeuaviot

3

tocaF

tep65bfeaiHao

TE

negatively charged), resulting in the decrease of EAC.As shown in Fig. 4(a), the optimal operation condition for endo-

toxin elimination is located at ionic strength of 0.15–0.30 mol/L

0.550.500.450.400.350.300.250.200.150.100.050.000.10

0.15

0.20

0.25

0.30

0.35

0.4010987654

Ionic strengt h

EA

C (E

U/c

m2 )

Ion ic stren gth (mol/L )

pH

pH

(a)

1.5

2.0

2.5

3.0

orbe

d en

toto

xin

(EU

)

(b)

PVDF membrane 286.519 46.9 290.915 31

Affinity membrane 287.063 39.7 291.021 2.4

riginal and affinity membranes. As shown in Table 2, the adsorp-ion capacity to BSA and endotoxin of the original membrane areoth high, which is mainly attributed to the hydrophobic character-

stic of the PVDF material itself. By contrast, the adsorption capacityf the affinity membrane to BSA decreased to a significant loweralue of 0.002 mg/cm2, whereas the endotoxin adsorption capacityas still kept at 0.103 EU/cm2. The recovery ratio of BSA is about

9%, which is approximately equal to the reported endotoxin affin-ty adsorbents [32]. The low protein adsorption is mainly attributedo the hydrophilic affinity membrane surface. In addition, the elec-rostatic repulsion force between negative-charged Ser (pI 5.68)nd BSA (pI 4.8) is beneficial to lower the BSA adsorption capacityf Ser affinity membrane when the operating conditions are con-rolled at pH 6.5. The relatively high endotoxin adsorption capacityefinitely confirms the particular affinity between Ser and endo-oxin.

Comparing to the endotoxin adsorption capacity reported bythers [12,19], the adsorption value of PVDF-Ser membrane seemso be much lower. To a large extent, it can be attributed to the dif-erent operation conditions, especially the great difference in thenitial concentrations. In our experiment, the initial endotoxin con-entration was less than 2 EU/ml whereas the other two adsorptionxperiments for purification of the contaminated biological prod-cts were taken at the concentration of 116.4 EU/ml, 796.9 EU/ml,nd 6000 EU/ml, respectively. So the lower adsorption capacityalue of PVDF-Ser membrane was commensurate with the lowernitial endotoxin concentration. The effects of operation conditionsn the endotoxin adsorption were further discussed as shown inhe followings.

.2.2. Effects of operation conditions on the endotoxin adsorptionTo investigate the interaction between the ligand and endo-

oxin, it is necessary to learn the adsorption behavior under variousperation conditions including ionic strength, pH and initial con-entration. The protein-free solutions were adopted for endotoxindsorbing tests by a batchwise method. The results are shown inig. 4.

Fig. 4(a) shows the effect of ionic strength on endo-oxin adsorption. The affinity membrane exhibited the highestndotoxin-adsorbing activity at 0.25 mol/L. The LPS solutions wererepared with seven different ionic strengths at the similar pH (pH.5). Meanwhile endotoxin molecule (pKa = 1.3) and Ser ligands (pI.68) are both negatively charged, and the electrostatic repulsionetween them is unfavorable to the adsorption. The electrostaticorce is weakened when the ionic strength increases, which is ben-ficial for the formation of hydrogen bonds between endotoxinnd Ser ligand. Hence, the endotoxin adsorption capacity (EAC)

ncreased as the ionic strength increased from 0.05 to 0.25 mol/L.owever, when the ionic strength was higher than 0.25 mol/L, thectivity of endotoxin was depressed according to the limit-equationf Debye–Hückel [33], which led to the decrease of EAC.

able 2ndotoxin and BSA adsorption capacity of PVDF and affinity membrane.

Sample Endotoxin (EU/cm2) BSA (mg/cm2)

PVDF membrane 0.225 0.022Affinity membrane 0.103 0.002

284.800 8.5 – 0 – 0284.800 44.2 289.017 8.0 285.764 5.7

The pH value also influences the adsorption capacity of the affin-ity membranes significantly. With the increase of pH value, the EACincreased at the beginning and then decreased after pH 6, while themaximum EAC of 0.343 EU/cm2 was obtained as shown in Fig. 4(a).The influence of the group ionization degree has to be consideredsince the ionization of amino group reduces with the increase ofpH while the ionization of carbonyl and phosphate groups reduceswith the decrease of pH [34,35]. The variation of the group ioniza-tion degree can change the dipole–dipole interaction and dipoleinduced dipole interaction, thus the formation of hydrogen bondsbetween ligand and endotoxins are affected subsequently. WhenpH is higher than 5.8, the electrostatic repulsion force will weakenthe bonding between the ligand and endotoxin molecules (both

3.02.52.01.51.00.5

0.5

1.0Ads

Initial concentrations o f e ndoto xins (EU/ml)

Test 1 Test 2 Test 3

Fig. 4. (a) Effect of ionic strength and pH on EAC of affinity membranes. The ionicstrength study (pH 6.5, sodium chloride solutions) and the pH study (ionic strength0.25 mol/L) were all performed at room temperature. (b) Effect of initial concentra-tions on adsorbed entotoxin of affinity membranes (pH 6.5, room temperature).

M. Zhang et al. / Journal of Membrane Science 405– 406 (2012) 104– 112 109

Table 3Impact of affinity membrane on trace components in plasma.

Plasma components Before

adsorbed

After

adsorbed

References

Alanine aminotransferase 5 4 5~45 IU/L

Aspartate aminotransferase 14 11 5~35 IU/L

Alkaline phosphatase 418 361 30~110 IU/L

Glutamyltranspeptidase enzyme 71 58 5~50 IU/L

Total protein 59.5 50.6 63~82 g/L

Albumin 22.9 21.2 35~53 g/L

Total bilirubin 5.2 4.1 0~1.2 mg/dl

Direct bilirubin 4.8 3.9 0~0.4 mg/dl

Indirect bilirubin 0.4 0.2 0~0.8 mg/dl

Mg 1.04 0.86 0.6~1.1 mmol/L

P 2.01 1.81 0.8~1.6 mmol/L

Uric acid 7.7 6.8 3.5~8.3 mg/dl

Creatine kinase 6 5 39~275 IU/L

Lactate dehydrogenase 162 137 60~213 IU/L

K

Na

Cl

Ca

Carbon dioxide combining power

Urea nitrogen

Creatinine

4.21

143

110

1.88

11.9

81

2.5

99

310

3.46

120

90

1.44

8.5

67

2.0

78

3.5~5.3 mmol/L

134~147 mmol/L

98~109 mmol/L

2.2~2.7 mmol/L

23~33 mmol/L

7~20 mg/dl

0.7~1.3 mg/dl

75~105 mg/dlGlucose

abaaa

saTms

q

weaw

Osmotic pressure

nd pH of 5–8. These two ranges are consistent with human bloodehavior, of which the ionic strength is approximately 0.15 mol/Lnd pH is around 7. Therefore, the affinity membrane can maintain

high endotoxin adsorption capacity in real human blood system,s will be shown in Section 3.3.

The adsorption isotherm of endotoxin is presented in Fig. 4(b),howing a linear positive correlation between the quantity ofdsorbed endotoxin and the initial concentration of LPS solutions.he experimental data indicates that the Freundlich adsorptionodel can be applied to describe the adsorption equillibria in this

ystem, which is expressed as:

∗ = KF (C∗)1/n (2)

here q* (EU) is the adsorption capacity, C* (EU/ml) represents thequilibrium concentration of endotoxins in solutions, and KF and nre the physical constants of Freundlich adsorption isotherm. In thisork, KF and n are calculated to be 0.838 and 0.936, respectively.

261 280~320 osmo/kg

In real hemoperfusion treatment, the temperature is usuallymaintained around 37 ◦C, the normal body temperature, so theKF and n values may vary in that temperature range. In relatedstudies about endotoxin-affinity adsorbent, removal efficiency wasreported to increase with rising temperature, due to the increasedavailability of the active sites for endotoxin binding induced by theincreased Brownian movement of endotoxin molecules [36]. How-ever, further adsorption thermodynamics research is necessary toconfirm how temperature rise acts on ET adsorption.

Generally, Freundlich model is developed to describe the mono-layer and heterogeneous adsorption. It assumes that the adsorptionenergy of binding a target molecule to a site depends on whetherthe adjacent sites are occupied [37–39]. Therefore, the adsorp-

tion of endotoxin on PVDF-Ser affinity membrane is supposed tobe a monolayer and heterogeneous type. The value of n in thissystem is close to 1, revealing clearly that there are positive coop-erative interactions between the endotoxin molecules adsorbed

110 M. Zhang et al. / Journal of Membrane S

50403020100

0.0

0.1

0.2

0.3

0.4

End

otox

in c

once

ntra

tion

of o

utle

t flo

w (E

U/m

l)

os

3

rTvpWwtafbtocfAfi

w

Volume of outlet flow (ml)

Fig. 5. Breakthrough curve of endotoxin concentrations to volume.

n affinity membrane and the free endotoxin molecules in theolution.

.3. Endotoxin removal from human plasma

The performance of PVDF-Ser affinity membrane for endotoxinsemoval from human plasma is evaluated by in vitro experiments.he breakthrough curve of endotoxin concentrations to outletolume is shown as Fig. 5. It suggests that the affinity membrane-acked module could reduce the level of endotoxin effectively.hen the original concentration was 0.42 EU/ml and the flow rateas 1 ml/min, the EAC of affinity membrane was 0.058 EU/cm2. Fur-

hermore, the endotoxin removal efficiency was determined to belmost 100% for 15 ml sample, 93.5% for 20 ml sample and 48.3%or the whole 40 ml sample. As also shown in Fig. 5, the mem-ranes hardly absorbed extra endotoxins after 33 ml plasma passedhrough the module, which was probably because most active sitesn the surface were saturated. Therefore, the membrane modulean be improved by optimizing the membrane preparation methodor higher grafting-serine density to get higher clearance efficiency.

greater purified volume can also be expected by increasing theber number or narrowing the fiber diameter.

The effect of adsorption process on the blood bio-chemistryas further evaluated by testing the plasma sample after a single

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filtration. The results are presented in Table 3. Considering theplasma sample is offered by the sepsis patient, it is reasonable thatsome values are not in the range of references. Comparing the pro-tein data in the original solution and the final solution, as shown inthe red-dotted frame in Table 3, the recovery ratio of total proteinwas calculated to be 85.1% and the recovery ratio of albuminwas 92.4%. In addition to albumin, the loss of total protein camefrom the globulin, which was considered to be related to immunefunction. For instance, IgG and IgM act to neutralize endotoxinand increase serum bactericidal activity, so the consumption ofantibodies may cause negative influence during sepsis. However,just as this conclusion remains controversial whether low blood Igconcentration is associated with a poorer outcome in patients withsepsis [40], it is difficult to tell the potential effect of reduced glob-ulin level in a real hemoperfusion treatment process, especiallyconsidering the complexity of immune response on single sepsispatient. Moreover, no significant deleterious effect was observedafter the adsorbing process. The PVDF-Ser affinity membrane hasdemonstrated its good potential in clinical application.

3.4. Hemoperfusion experiment in pig model

As aforementioned, it was confirmed that endotoxins could beremoved from human plasma in vitro by PVDF-Ser hollow fiberseffectively. The further animal experiments were finally investi-gated to check whether it is feasible to apply the hollow fiberaffinity membrane module in the direct hemoperfusion treatment.

The plasma endotoxin, TNF-� and IL-6 concentrations of bothhemoperfusion (HP) group and control group were tested every 1 hduring the endotoxin infusion period and the following 3 h. Thedata are shown in Fig. 6. The endotoxin level increased within thefirst 2 h and then began to decrease, while inflammatory mediatorlevels reached their peaks 3 h after the endotoxin infusion started.The HP group exhibited lower final values than the control groupat the end of the experiment.

At the end of infusion, the endotoxin concentrations of HPgroup and control group both increased to nearly ten-fold of theoriginal level, but during the following 3 h, HP group had moresignificant decrease, especially at the initial 2 h of hemoperfu-sion treatment. The final endotoxin concentration of HP group

decreased to 0.022 EU/ml. Moreover, the endotoxin removal effi-ciency was calculated to be 70% for control group and 93% for HPgroup. It indicates that although the endotoxin reduction is partiallyowing to the immune responses of pig’s own immune system, the

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VDF-Ser adsorber is still proved to be effective for the eliminationf endotoxins.

For TNF-� and IL-6 results, the levels kept increasing duringhe first 3 h, demonstrating that these two inflammation mediatorsere released even after endotoxin infusion. In the following 1 h,

he TNF-� and IL-6 levels of HP group began to decrease much moreemarkably than control group. Since these two mediators are nor-ally involved with the systemic inflammation responses which

re induced by endotoxins exposure, the decrease of them revealshe relief of inflammatory reaction, and indicates the endotoxineduction.

. Conclusions

The l-Ser can successfully be grafted onto the surface of PVDFollow fiber membrane by the proposed hydrophilic modificationethod, and the ligand density obtained is 4.42 �mol/cm2. The

urface-bound Ser allows the matrix to selectively adsorb endo-oxin from protein solution.

The adsorption experiments prove that PVDF-Ser adsorbentaintains high adsorption capacity in the ionic strength range of

.15–0.30 mol/L and the pH range of pH 5–8, which exactly fits theperating conditions of blood treating. In this work, the adsorptionf endotoxin is found to be monolayer and heterogeneous.

Additionally, adsorber packed with PVDF-Ser fibers can effec-ively remove endotoxin from human plasma. The endotoxindsorption capability (EAC) is 0.027 EU/cm2 and the removal effi-iency reaches almost 100% for 15 ml sample and 48.3% for 40 mlample at controlled initial concentration and flow rate. The bio-hemical test shows that the adsorber induces no deleterious effectn plasma when the recovery ratio of total protein is 85.1% and theecovery ratio of albumin is 92.4%.

The therapeutic efficacy of the scaled-up adsorber is confirmedy extracorporeal hemoperfusion treatment on septic pigs. Thedsorber could decrease the levels of plasma endotoxin, TNF-� andL-6, indicating that blood purification using this novel PVDF-Serdsorber may be a potentially promising way in sepsis therapy.

cknowledgments

The authors gratefully acknowledge the financial support pro-ided by the Key Scientific and Technological Project of Sciencend Technology Department of Zhejiang Province, China (No.007C33008), and Ministry of Education, Taiwan. We also appre-iate Sir Run Run Shaw Hospital, School of Medicine, Zhejiangniversity for the assistance with animal experiment.

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