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Biomaterials 32(2011) 352-365

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Biomaterials

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The biocompatibility and separation performance of antioxidative polysulfone/vitamin E TPGS composite hollow fiber membranes

Ganpat J. Dahe, Rohit S. Teotia, Sachin S. IZadam, Jayesh R. Bellare*Deportment of Chemical Engineering, Indian Institute of Technology Bombay. Powoi. Mumboi - 400 076, lndia

A R T I C L E I N F O A B S T R A C T

Article historyrReceived 13 August 2010Accepted 2 September 2010Available online 2 October 2010

Keywords:Hollow fibersTPGS

BiocompatibilityUltrafiltration coefficientSolute rejection

The extended interaction of blood with certain materials like hemodialysis membranes results in theactivation of cellular element as well as inflammatoy response. This results in hypersensitive reactionsand increased reactive oxygen species, which occurs during or immediately after dialysis. Althoughpolysulfone (Psf) hollow fiber has been commercially used for acute and chronic hemodialysis. itsbiocompatibility remains a major concern. To overcome this. w e have successfully made composite Psfhollow fiber membrane consisting ofhydrophilic/hydrophobicmicro-domains of Psf and Vitamin E TPGS(TPGS). These were prepared by dry-wet spinning using 5.10.15.20 wt%TPGS as an additive in dopesolution. TPGS was successfully entrapped in Psf hollow fiber, as confirmed by ATR-FTIR and TGA. Theselective skin was formed a t inner side of hollow fibers, as confirmed by SEM study. In vitro biocom-patibility and performance of the PsflTPGS composite membranes were examined, with cytotoxicity. ROSgeneration, hemolysis, platelet adhesion. contact and complement activation, protein adsorption.ultrafiltration coefficient, solute rejection and urea clearance. We show that antioxidative composite Psfexhibits enhanced biocompatibility,and the membranes show high flux and high urea clearance, abouttwo orders of magnitude better than commercial hemodialysis membranes on a unit area basis.

O 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Hemodialysis is avital clinical process for removal of toxins such ascreatinine. urea. biological metabolites and free water from blood in

renal failure. The core element of a hemodialysis is ultrafiltration

hollow fiber m embr ane (HFM), which selectively permits toxins from

blood via diffusive and convective transport across th e membrane.Polysulfone (Psf) hemodialyzer are widely used due to excellentmembrane formation ability. chemical inertness, mechanical strength.

and thermal stability, which make it one of th e few biomaterials thatcan withstand sterilization techniques. Despite the popularity of

membrane material. the biocompatibility is still a major concern. Thecontact of blood proteins and cells with HFM surface activates

inflamm atoy response (coagulation,fibrinolysis, complement cascadeand kallikrein-ltinin) and cellular element (platelet, neutrophils,monocytes, hemoglobin release through erythrocyte rupture) [I-61.

Earlier studies demonstrated platelet activation, contact acti-

vation and increased cytokine production on Psf hemodialyzer[7-91. To improve biocompatibility of Psf, researchers have

* Corresponding author. Tel.: +91 22 2576 7207;fax: +91 22 2572 6895.E-mail address: jb@iitb.ac.in (J.R. Bellare).

modified me mbran e surfaces using different methods . including:

air-plasma glow discharge treatmen t, graft copolymer preparation

ofpolysulfone-g-poly(ethy1eneglycol) (Psf-g-PEG)and its blendingwith Psf [10.11]. The most widely used method for improvingbiocompatibility of Psf membranes is the use of additives having

excellent biocompatibility than the native polymer. Psf blended

with polyvinylpyrrolidone (PVP) showed enhanced biocompati-bility than native Psf. PVP increases the hydrophilicity of the

membrane surface and also acts as a pore forming agent [12]. Ish-ihara et al. 1131 prepared a phospholipid polymer having a 2-methacyloyloxyethyl phosphoylcholine (MPC) unit. The MPC

polymer wa s blended wi th Psf by solvent evaporation method. The

platelet adhesion and protein adsorption were reduced and change

in morphology of adherent platelets was suppressed.

Another critical issue of Psf hemodialyzer is oxidative stress

produced by reactive oxygen species (ROS) during hemodialysis[14.15]. ROS are largely produced by neutrophils and monocytethrough protein and lipid oxidation [16]. Increased ROS are th oughtto be involved in atherosclerosis, hypertension or chronic inflam-

matory diseases and nephritis [17.18]. Prelim inary studies employ-ing the Psf membrane a nd antioxidant agent such as vitamin E have

showed significant improveme nt in neutrophil function. hematocrit

and qua lity of life [19-211. Sasaki et al.. prepared vi tamin E modified0142-9612116- see front matter O 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.biomaterials.2010.09.005

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G.J.Dahe er al. / Biomatenols32 (2011) 352-365 355

3. Result and discussions

3.7. Characterization of hollowJber membinne

3.1.1. Morphology study by SEMSEM cross section of prepared P, PT-5. PT-10. PT-15 and PT-20

HFMs demonstrated uniform wall thickness along the circumfer-

ence with finger like macrovoids, as shown in Fig. 1. The wall

thickness of native Psf is smaller than composite Psf membranes.

The elongation of native Psf fiber in air gap is more compared to

composite Psf fiber due to its lower dope viscosity.TPGS addition to

dope solution increases its viscosity, which is responsible for

increase in wall thickness of composite Psf HFMs.

These experimental membranes have inner diameter and wallthickness of about 600-650 pm and 150-180 pm respectively,which are approximately three times larger than commercialhemodialysis membranes. This is a constraint of the experimental

fiber spinning line we have. The HFMs with equivalent dimensions

to commercial fiber with same performance can be prepared by just

replacing spinneret with smaller dimension, which is planned forthe future. The objective of this study is to first develop anti-oxidative HFMs with improved biocompatibility and separation

performance, so we have showed the comparison on a per unit area

basis.The prepared HFMs showed dense layer formation (membrane

skin) at inner side and porous structure at outer side for all fibers

(typical micrographs shown in Fig. 2). This is an essential micro-structure required for hemodialysis membranes (391. The innersurfaces of HFMs were not observed under SEM due to its fine

structure.

3.1.2. Surface composition analysisby ATR-FTIRThe presence of TPGS at inner surface of HFM was confirmed by

ATR-FTIR spectroscopy. ATR-FTIR spectra of Psf and composite Psf

membranes are shown in Fig. 3a. All ATR-ITIR spectra showed moststrong bands in the "finger-print" region of spectrum below1700 cm-I. The Psf consists of a backbone made up ofdiaryl sulfone(Ar-SO2-Ar) and diaryl ether (Ar-0-Ar) groups showingpronounced strong absorption peaks at 1151 and 1242 cm-'respectively. The band at 1488, and 1586 cm-' belongs toCH3-C-CH3 stretching and vibration of the aromatic C= C in Psfmolecule respectively. Beside these, there are two more strongabsorption bands associated with vibration of the sulfone group.

appeared a t 1294 and 1324cm- he presence ofTPGS in cornposite

Fi g1. SEM micrographs of cross section of whole HFMs (a ) P. (b) PT-5.(c ) P-TIO.(d) PT-15 and (e) PT-20 [scale bar: 500 ~ r n ]

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GJ.Dahe et al. / Biomatenals 32 (2011) 352-365 357HFMs performance. Kim and Lee observed similar findings for

polyethylene glycol (PEG) as additive in Psf membranes 1401.

3.2. Mechanism of TPGS entrapment in HFMs membranes

HFMs are prepared using phase inversion technique. The

precipitation occurred through mass transfer between solvent and

nonsolvent. The selective layer (skin) was formed at polymer sol-ution-nonsolvent (bore solution in case of HFM) interface followedby porous structure i.e. lacy and open cellular. Wienk et al.. reportedpacked nodular structure of ultrafiltration membrane skin [41].Thesmaller nodules are present at surface and nodule size increasesdeeper into the film until it gradually transforms into a porous

structure.The polymer solution consists of Psf(M, 79.000) and TPGS (Mw

1513) in N-methylpyrrolidone (NMP). The rapid diffusion of the

water (nonsolvent) in polymer solution and out diffusion of NMP

(solvent) confronts an incompatible environment specifically to Psf

molecules. These polymer molecules (Psf and TPGS) are an highlyentangled coil due to high polymer concentration. Psf molecules

reduce its interactions with the nonsolvent by clustering thepolymer segments of both Psf and TPGS into groups. TPGS is

amphiphilic and compatible with Psf and water. The polymerclusters/nodules may consist of polymer segments of Psf and TPGS.and adjacent clusters are connected by entanglements or sharing

Psf polymer molecules. The nodule size increases in direction ofnonsolvent diffusion as nonsolvent diffusion decreases along the

direction due to already formed polymer clusters. Thus polymermolecules get more time to form clusters and more number of

polymer molecules participates in cluster formation. During thetime, excess out diffusion of solvent increases the polymer

concentration of the top layer drastically, which results in vitrifi-

cation of the polymer matrix. This helps in holding TPGS molecules

by entangled and tightly packed Psf. The proposed mechanism isschematically represented in Fig. 4. TPGS molecules have inter-

molecular interaction by hydrogen bonding with Psf chains. TPGS

loading in Psf HFMs does not increase significantly with increasing

TPGS concentration in dope. The TPGS holding ability of cluster

could be saturated a t5%TPGS in dope. The excess TPGS moleculesdiffuse out easily due to its small size and compatibility with water.

This induces more porous structure in HFMs which ultimatelyinfluence the performance discussed in later section.

iNonsolvent-polymer solution interfaceFi g 4. Schematic diagram showing

Culture Period (Days )

Fig. 5. Cell viability assay ( M l T ) of N1H3T3 cells cultured o n P, m-5. FT-10, PT-15 andm-20 HFMs for 1 . 3 and 5 days.

3.3. Biocompatibility evaluation of hollowJber membrane3.3.1. Cytotoxicity study using NIH373 cell3.3.1.1. Cell proliferation. Cell viability ofNIH3T3 on the surface ofHFMs was evaluated at day 1.3 and 5 by MTT assay and the resultsare summarized in Fig. 5. Psf and composite Psf fibers exhibiteda similar growth pattern for NlH3T3 cells. Cell proliferationincreases with culturing time indicating proficiency of Psf and

composite Psf fibers to support the proliferation of NIH3T3 cells.Cells proliferation rate was found to be more on composite Psf

fibers than native Psf fibers indicating enhanced biocompatibility.

3.3.1.2. SEM study. When cells contact with biomaterial, theyundergo morphological changes to stabilize the cell biomaterial

interface. The cell adhesion and spreading consists of sequentialprogress of cell attachment. filopodial growth. cytoplasmic

webbing. flattening of the cell mass and fusing of cells to form mat.

These can be effectively studied using scanning electron micros-

copy (SEM) due to large depth of field, resolution and view area

'base Separation: Solvent-NonsolventDiffusion

mechanism of TPGS entrapment by phase separation

Networked Polymer Chain Clusters

holding TPGS

during the membran e formation.

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358 G.J.Dahe et al. / Biomatenals 32 (2011) 352-365compared to laser and optical microscopy [42].On day 5, outersurface of Psf and composite Psf fibers showed NIH3T3 cellattachment with normal cell morphology (Fig. 6). The magnified

images clearly indicate cytoplasmic webbing of the cells and

extended filopodia on all HFMs surface. The cell monolayer wasobserved on the outer surface of composite Psf HFMs. Cell attach-

ment on outer surface of native Psf HFMs is also seen but t he time

taken to form the complete monolayer was comparatively longer.

These observations suggest t hat the composite membranes provide

better surface for cell attachment and proliferation.

On other hand, inner surface of composite and Psf HFMs does

not show any cell attachment; though the cells were observed at

planer cross sectional edges (Fig. 7). This may be due inability of

cells to attach on concave surface. In static condition almost no cells

were attached. which means in flowing condition chances of cell

adhesion are critically low.

3.3.1.3.Confocal study. Fluorescent-tagged phalloidin specificallystains the actin skeleton in cells. The cell adhesion on outer surfaceof Psf and composite Psf HFMs at day 5 was studied using confocalmicroscopy. Profuse cell growth was observed in all composite Psf

HFMs (Fig. 8b, c, d and e). compared to native Psf HFM (Fig. 8a).These findings co-relate with MTT assay and SEMstudy. Cellmonolayer was observed on outer surface of HFMs with well-developed actin filaments and distinct rounded nuclei, indicatingnormal cell growth. No significant difference was found amongdifferent composite Psf HFMs. Significant cell adhesion and growth

was observed in composite Psf HFMs compared to native Psf HFM.

indicating increased biocompatibility. Increased cell proliferation in

composite Psf membranes is due to presence of vitamin E in

conjunction with PEG. Vitamin E part of TPGS suppresses ROS

formation, which helps in reducing aging ofNlH3T3 cells and inturn increases cell proliferation.

3.3.2. OxidativestressOxidative stress is vital indicator for induced cellular toxicity

[43.44]. ROS induced cell apoptosis is well evidenced [45]. Theregulation of oxidative stress is a logical mean of tuning the bio-

logical response to materials. 2',7'-Dichlorofluorescin diacetate(DCFH-DA) fluorescence was used as a marker of oxidative stress in

the cells. DCFH-DA enters inside cell and converted to 2',7'-dichlorodihydrofluoresceine (DCFH) by cellular esterase activity.

Fig. 6. SEM micrographs ofNIH3T3 cells showing cell attachment on outer surfaces of (a) P, (b) PT-5, (c ) P-TIO. (d) PT-15 and (e) PT-20 HFMs at day 5 [scale bar: 200 pm (inset:50 V ) l .

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GJ. Dahe et al. / Biornotenals32 (2011) 352-365 359

F i g 7. SEM micrographs ofNIH3T3 cells cultured on inner surface (a) P (b) PT-5. (c) P-TIO,(d ) PT-15 and (e) PT-20 HfMs showing almost no cell attachment at day 5 [scale bar:200 pm (inset: 100 pm)].

Low molecular weight peroxides produced by the cells oxidize DCFH

to highly fluorescent compound 2'. 7'-dichlorofluorescein (DCF).

which remain trapped within the cells [46].Fluorescence intensity isproportional to the amount of peroxide produced by the cells. DCF

fluorescence was found to be maximum for native PsfHFM at day 1.3and 5 (Fig. 9). The DCF fluorescent intensity steeply increased fornative Psf and Hemoflow F6 HFMs with cell incubation period. Thecomposite Psf HFMs showed significant decrease in DCF fluorescent

intensity with respect to native Psf and Hemoflow F6 HFMs. While,no significant difference was observed among the composite PT-5,

PT-10, PT-15 and PT-20 membranes. The results showed reduction inROS generation in case of composite Psf HFMs due to incorporation

of TPGS. The D-a-tocopherol (Vitamin E) content in TPGS is 25%.The

antioxidative function of vitamin E as a peroxyl radical scavenger iswell studied 147,481.This indicates that vitamin E content in TPGS isresponsible for reduction in oxidative stress in NIH3T3 cells andconfirms antioxidative property of composite Psf HFMs.

3.3.3. Protein adsorptionWhen blood is in contact with biomaterial. the initial event is

adsorption of plasma protein on its surface. This is the basis of

platelet adhesion and activation of coagulation pathways, leading

to thrombus formation I49.501. Hence. protein adsorption isessential for evaluation of biocompatibility. Fig. 1Oa showsadsorption of human plasma proteins (albumin. y-globulin and

fibrinogen) on the three membranes studies, namely native Psf,

composite Psf and Hemoflow F6, where it can be seen that theamount of albumin, y-globulin and fibrinogen adsorbed on native

Psf (control. taken as 100%)and composite Psf HFMs (73 .60.48 and50%for PT-5, PT-10, PT-15 and PT-20 respectively) are lower thanthe Hemoflow F6 (135%).The undesirably higher value of proteinadsorption on the Hemoflow F6 is not possible to explain since it isa Psf membrane with undisclosed additives. but it is apparent that

the TPGS additive to Psf significantly helps improve (lower) the

adsorption on our composite Psf HFMs.

For the protein adsorption studies. the protein concentrations were

formulated to resemble physiological concentrations, so the concen-

tration of albumin and y-globulin used is much higher than the

fibrinogen. Despite this. the amount offibrinogen adsorbed was about

the same as albumin and y-globulin.This may be due to greateraffinityof fibrinogen to membrane surface, because of the large net negative

charge of fibrinogen as compared to albumin and y-

globulin 1491.

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360 GJ Dahe et aL / Biomateriak 32 (2011)352-365

Rg.8. Confocal laser micrographs ofNIH3l3 cells cultured on (a) P. (b) PT-5. (c) PT-10. (d )PT-15 and (e)PT-20 stained with HTC-phalloidin for actin filaments (green) and DAPl fornuclei (blue) [scale bar: 100 pm (inset: 20 pm)].(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The adsorbed quantity of albumin, y-globulin and fibrinogen

gradually decreases with increasing TPGS concentration. attr ibuting

the influence of TPGS additive. TPGS contain hydrophilic PEG

segm ent, which makes t he surface hydrophilic. It is observed th at

PEG immobilized PET surfaces lower protein adsorption [51]. Thehydrophilicity of surface is responsible for lowering the protein

adsorption. Similar results were obtained w hen Psf memb rane wer e

coated with pluronicTMwit h increasing order of hydrophilicity 1521.

3.3.4.Hernocompatibility test3.3.4.1. Hemolysis. In hemodialysis , blood continuously flows

through th e lumen of the hollow fiber. Blood normally consists of

48%erythrocyte volume fraction. Interaction of erythrocytes wi th

hollow fiber is essential to st udy t he release of hemoglobin (called

hemolysis). In vitro hemolysis test was performed on native.

composite Psf and Hemoflow F6 HFMs. The percentage hemolysisoccurred in native. composite Psf and Hemoflow F6. when

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352 GJ. Dahe et al. / Biomoteiials 32 (2011) 352-365

Fig11. SEMmicrograph of platelets adhered on inner surface of (a) P,(b ) PT-5. (c) P-TIO. (d) PT-15. (e ) PT-20 and ( 0 Hemoflow F6 HFMs [scalebar: 50 pm (inset: (a)-10 pm: (b),(c).(d l , (e l and (0 -2 11m)l.

which may be the reason for increasing Itallikrein activity.Composite Psf HFMs showed significantly lower kalliltrein activity

compared to native Psf, may be due to reduced fibrinogen adsorp-

tion. Sasaki found reduction in activation of contact phase for

vitamin E coated Psf HFM compared to native Psf [24].3.3.6. Complement activation

Complement activation is the triggering of host defence mech-

anism by generation of localized inflammatory mediator. Hemo-dialysis membrane showed activation of complement by

alternative pathway [S].Complement activation is measured bydetermining the generated anaphylatoxins C3a. C4a and C5a orterminal complement complex (TCC). Among all, TCC is a superior

index of biocompatibility due to stable terminal complex formation

generated by classical, lectin or alternative pathway 1581.Therefore,TCC concentration was determined using enzyme linked immu-nosorbent assay (ELISA) for complement activation study by HFMs.

TCC concentration was maximum for native Psf HFM (2052.0 ng/ml). while PT-20 (604.16 nglml) and Hemoflow F6 (764.6 nglml)showed minimum (Fig. 12b). This manifests generation of acuteinflammatory response of native Psf HFM demonstrating poor

biocompatibility. PT-20 membranes exhibited considerably lower

complement activation among the native. composite Psf andHemoflow F6 HFMs. This could be due to poor binding of C3 frag-ments to the composite HFMs due to presence of TPGS. Identical

trend was observed in case of cellulose acetate and vitamin E

modified cellulose (Excebrane E) membrane. where TCC concen-

tration was 990 ng/ml and 340 ng/ml respectively [23].3.4. Performance of the hollowfiber membranes3.4.1. Ultrafiltration coelficient (KuF)

Ultrafiltration coefficient is the measure of efficiency of dialyzer.

The diffusion of solutes like urea creatinine, glucose is dependenton ultrafiltration coefficient. The bar plot of prepared native,

composite and Hemoflow F6 HFMs is shown in Fig. 13a. KUF ofcommercial (Hemoflow F6) is 4.76 ml/h m2 mm Hg. while I(UF ofcomposite HFMs increases from 16 to 54 ml /m

2hr mm of Hg with

TPGS addition. It is reported that the pore numbers and porosity

increase with low molecular weight additive like PEG in dope 1401.These can be proved from TGA studies. TPGS content in final

composite HFMs ranges from 6 to 8 wt%,though the TPGS was

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GJ. Dahe et al. / Biomatenals 32 (2011) 352-365

" P PT-5 PT-10 PT-15 PT-20 HemoflowTime (minutes) F6Membrane Type

fig12. (a) Kallikrein activity of plasma after contact with Psf, composite Psf and Hemoflow F6 HFMs for 30 min detected by cleavage of chromogenic substrate (5-2302) and (b)Concentration of SC5b-9 in incubated plasma with Psf, composite Psf and Hemoflow F6 HFMs for 30 min (Data are expressed as the mean i SD of three independentmeasurements).

increased from 17.67 to 44.50 wt%with respect to Psf. This signifiesthat TPGS leaching increased from 11.67 to 36.5 wt%,which leads tomore porous structure formation. thereby increasing lZuF TheCenters for Disease Control (US) defined high-flux dialyzers withultrafiltration coefficients of 20 ml/hr m2 mm of Hg or greater [59].The membrane prepared with TPGS additives enhanced KU~ ;showing characteristics of high-flux dialyzer. It has been clinically

reported that better survival was observed, when high-flux

hemodialysis membranes were used in patients for a long period

[60]. High flux membranes have a comparatively low diffusiveresistance and greater clearance of larger solutes such as vitamin

BI2 (1355 Da). Thus. membrane prepared with 20%TPGS additiveshows highest ultrafiltration coefficient. far superior than allmembranes studied here including the commercial Hemoflow F6membrane.

Membrane Types

3.4.2. Solute rejectionThe solute rejection of prepared HFMs and Hemoflow F6 is

shown in Fig. 13b. The solute rejection curves of native andcomposite Psf HFMs almost overlaps with Hemoflow F6. indicatingequivalent performance as far as solute rejection is concerned. The

solute rejection is governed by pore size and pore size distribution.Therefore, pore size and its distribution of prepared HFMs are

similar to that ofHemoflow F6. Nominal molecular weight cut off(NMWCO) is th e molecular weight of solute corresponding to 90%

rejection. The NMWCO of native. composite Psf and Hemoflow F6HFMs varied from 4300 to 5000 Da. This indicates no significant

difference in NMWCO among the native, composite Psf andHemoflow F6 HFMs. The MWCO of prepared native, composite andHemoflow F6 HFMs imply no loss of albumin from blood. Thecomposite PsfHFMs having same solute rejection profiles to that of

1000 10000Molecular Weight (gmlmol)

F i g13. (a ) Bar plot of ultrafiltration coefficient of native Psf, composite Psf and Hemoflow F6 HFMs and ( b) Solute rejection plot of native Psf, composite Psf and Hemoflow F6HFMsmeasured by gel permeation chromatography.

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