Preparation and properties of plasma-initiated graft copolymerized membranes for blood plasma separation

Radtat Phys Inr J Radial

Chem Appl

Vol 39, No 6, pp Instrum, Part C

F’nntcdm Great Bntam All n&s

569-576,

1992

OM-5724/92 $5 00 + 0 00 1992 PcrgamonF?cssLtd

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PREPARATION AND PROPERTIES OF PLASMA-INITIATED GRAFT COPOLYMERIZED MEMBRANES FOR BLOOD PLASMA SEPARATION M. ONISHI,‘~ K. SHIMURA,’ Y. SEITA,’S. YAMASHITA,’A. TAKAI-LWII’and T. MASUOKA~ ‘R&D Center, Terumo Corp , Inokuchll500, Nakai-machl, Kanagawa 259-01, Japan and 2Research Institute for Polymers and Textiles, l-l-4 Higashi, Tsukuba-shl, Ibarakl305, Japan Abstract-A

hydrophlhc composite membrane for blood plasma separation has been pnpared by surface graft copolymenzatlon mltlated by low-temperature plasma (LTP). After short LTP prekradiation onto a mlcroporous polypropylene (PP) membrane, N-N-dlmethylacrylanude (DMAA) vapor was mtroduced for grafting The PP membrane had a 0 45 pm effective pore size and a 130gm tickness The rate of DMAA grafting onto PP was very high, even m vapor-solid phase reaction under reduced pressure, DMAA 1 mm Hg (133Pa) The percentage of grafted poly-DMAA (PDMAA) reached 15% Hnthm 5 rmn post graft polymenzation, and the membrane surface, including the interior surface of pores, became completely hydrophdlc There was no apparent change observed m the membrane morphology m the dry state after the PDMAA-grafted layer was formed However, water flux slgmficantly decreased, probably due to swelling of the PDMAA-grafted layer With a grafting yield below 17%, the PDMAA-grafted PP (PP-g-PDMAA) membrane showed a good separation capahhty of plasma from whole blood. The PP-g-PDMAA membrane exhibited low complement actlvatmg potential, Hugh slevmg coefficient for plasma proteins and lugh blood compatiblhty Decreases m adsorption of blood cells, plasma protems, and other blomolecules may be the reason for the membrane performance.

INTRODUCTION

plasma (LTP) has been utilized for surface modlficatlon of polymenc matenals for about twenty years already (Yasuda, 1986, 1981; Clerk, 1978; Hallahan and Bell, 1974) There are three types of LTP processmg: simple LTP treatment, LTP polymenzatlon, and LTP-initiated graft copolymenzatlon (LTP-graft). Many authors have reported that only the surface layer of a polymeric material can be modified by LTP and that little changes can be observed m the bulk repon of treated materials. Therefore detenoratlon of the mechanical strength 1s mlmmized. The reason is because the action of LTP 1s relatively mild and it does not penetrate materials as much as other lomzing radiation, such as gamma rays or electron beams, and others do (Mimura et al., 1978). It has been thought that LTP does not easily expand into small tubes, narrow gaps and trenches of matenals. However, the influence of LTP irradiation m microporous matenals has not been clearly investigated. LTP contains fast electrons, radicals, ions, and reactive metastable species. These species may contribute to the modification of the material surface together with energetic light enussion in the vacuum ultraviolet region (wave length < 200 nm) from LTP. One of the most remarkable effects is to form radicals on polymer substrates; radicals that can initiate crosslinking, chain scission, and graft copolymerization

Low-temperature

tTo whom correspondence

should be addressed.

(Corbm et al, 1985; Fales et al., 1976; Moshonov and Avny, 1980) If the radicals are generated deep inside a porous substrate by LTP-irradiation, the interior surface of pores wdl be modified by LTP-grafting. Ongmally we started this work to modify the interior of porous membranes (Masuoka et al., 1986). If LTP can modify a porous substrate deep inside as well as on the outer surface, the application area of LTP treatment, or of mlcroporous membranes may be further expanded. Hydrophlhc polymenc membranes are of interest to the blomedlcal mdustry for their numerous applications ranging from extracorporeal blood treatment to separation and punfication of biomolecules. Porous PP is a hydrophobic material. This means that aqueous media such as water and blood can not penetrate the pores, unless a positive pressu:e gradient is applied. Porous PP can be converted to a hydrophilic membrane by treatment mth surfactants. However, the surfactant-treated porous PP is unsmtable for applications in which it has to get in contact with blood or tissues because the surfactant leaches out from the membrane matnx. In a previous study we demonstrated that the intenor of a rmcroporous substrate could be modified by LTP-graft (Masuoka et al., 1989). In the present work, m order to prepare a hydrophilic composite membrane for biomedical application, LTP-graft of N-Ndimethylacrylamide (DMAA) onto microporous polypropylene (PP) membrane was studied. DMAA is a hydrophilic monomer, and its polymer (PDMAA) is watersoluble. Therefore, grafted PDMAA chains, which

M ONISHIet al

570

are covalently bonded to the PP surface, would produce a highly hydrophrhc layer The arm of this study was to prepare drmensronally stable, permanently hydrophrhc, and blood compatrble membranes for such biomedical applications as blood plasma separation Recently, membrane technology 1s being mcreasmgly applied to the collectron of plasma by donorplasmapheresrs, a procedure m which plasma 1s separated from whole blood and the cellular components are reinfused back to the donor (Jaffrm, 1989; Stromberg et al, 1987) Some membrane-based donor plasmapheresrs systems are currently m use (Stromberg et al, 1989) However, a few problems concerning blood compatrbrhty are still discussed, for example complement activatton (Omokawa et al, 1989) An evaluatron of PP-g-PDMAA membrane as a plasma separation membrane 1s also presented. EXPERIMENTAL

Preparation of grafted membranes A mrcroporous PP membrane with an effective pore size of 0 45 pm and a thickness of 130 pm was prepared by the thermally-induced phase separation method from a mixture of PP, hqurd paraffin and a nucleating agent (Serta and Omshr, 1988) The monomers for grafting, N,N-drmethylacrylamrde (DMAA) (KoJm Co , Japan) and ethylacrylate (EA) (Kant0 Chemical Co, Japan) were punfied by vacuum dtstillatron and degased by freeze-thaw cycles under reduced pressure The PP substrate, supported by a stainless steel mesh, was placed m the center of the reaction vessel, and was exposed to argon LTP that was excited by a 13.56 MHz radio frequency m the absence of graft-monomer After LTP rrradratron, the vessel was immediately evacuated to lo-’ mm Hg (0 13 Pa), and monomer vapor was mtroduced mto the vessel to mrtrate graft copolymenzatron The resultmg membranes were Soxhlet extracted with methanol for 48 h to remove non-grafted DMAA and PDMAA Properties of grafted membranes The degree of grafting (%G) was calculated from the weight of grafted (W,) and non-grafted (W,,) samples by the followmg equation %G = 100 x ( WB- W,)/ W,, Wettabthty was examined by placing the porous membranes on the surface of water and measurmg the time reqmred for the filters to become completely wet (ASTM-D4199 method B). Water flux measurement was camed out under a pressure of 0 7 kg/cm’ using an ultrafiltratron cell, model UHP-47 (Advantee Toyo Co., Japan). The bubble point was determined with isopropyl alcohol m accordance with the method defined by ASTM-F316. X-Ray photoelectron spectra (XPS) were measured by the JEOL 90SX to estimate surface coverage with

the grafted polymer. Scanning electron mmrograph (SEM) and nuclear magnetrc resonance (NMR) spectra were obtained by using JEOL JSM-840 and JEOL-FX30A, respectively FT-IR-ATR (Attenuated total reflectance Fourier transform infrared spectroscopy) was DIGILAB FTS-40 Platelet adhesron test The platelet adhesion test was camed out using sodium citrate added platelet rich plasma (PRP) freshly prepared from the blood of a healthy person PRP was dropped on the surface of non-porous films. After 30 mm mcubatron at 298 K, the films were rmsed wrth saline solution, and were treated by fixation wrth 1% of glutaraldehyde phosphate buffered solutton for 16 h at 277 K The amount and shape change of platelets adhering on the surface of the films were mvestrgated by SEM observation The adhered platelets were classrfied mto 3 types, type 1 to type III, according to their morphologrcal changes (Yonaha et al., 1980) Plasma separation experiment A small test module (effectrve area, 24cm2, blood flow path thickness, 350 pm) was used to measure plasma filtration flux from fresh bovine blood treated with heparm and ACD The wall shear rate was raised stepwrse every 10 mm at 150, 300, 500 s-l, and the plasma filtrate (Qr) was measured at 37°C. Sieving coefficients (Sc) of total protein, albumin and total cholesterol were calculated from the concentration of inlet blood (C,,) and filtrated plasma (C,) by the following equation

The complement-activatmg potential of the polymenc membrane was evaluated by C3a, C4a, and C5a production durmg rn vrtro plasma separation, using fresh human blood treated with ACD The experiment was performed with a small test module (effective area, 24cm2; blood flow path thickness, 100 pm) for 40 mm at room temperature (23-25°C) Blood flow rate (Qb) was kept at 0 5 ml/mm and plasma filtrate rate (Q,) was about 0.1 ml/mm C3a, C4a, and C5a were measured by radrormmunoassay (Amersham kit) The concentrations of F VIII C (KABI kit) and fibrinogen (Amencan Dade kit) were measured to investigate the vanatrons of the srevmg coefficient during m vitro plasma separation, usmg fresh human blood treated ACD The experiment was carried out with a disk type module (effective area, 116cm2, blood flow path thickness, 85 pm) at 37°C Qb and Q, were kept at 3 and 0.8 ml/min, respectively RESULTS AND DISCUSSION

Preparation

of the grafted membranes

The effect of LTP exposure-time on grafting yield (%G) is shown in Fig. 1. The percentage of grafted

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Fig 3 Dependence of maximum pore diameter on % grafting maximum pore diameter was calculated by the data from the bubble point of the membrane of the membranes was from 0 45 to 0 55 pm From these data, PP-g-PDMAA membranes seemed to retam a sufficient pore size for blood plasma separation Actually, there were no stgmficant morphological differences between the two membranes m the dry state when observed by SEM (Fig 4)

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Rg 5 Dependence of water flux on %G (0) PP-gPDMAA, (A) PP-g-PEA The water fluxes of grafted membranes are given m Fig 5 In case of PP-g-PDMAA membranes, though no apparent morphological difference was observed, the water flux decreased remarkably as the extent of grafting increased, probably due to swelling of the PDMAA-grafted layer on pore walls On the other hand, poly(ethylacrylate)-grafted-PP-g-PEA) membranes retained almost the same water permeabihty as the base membrane, because PEA is a relatively hydrophobic polymer and does not swell m water NMR is a powerful tool for charactenzation of polymers. However, not all the carbons m polymers contribute to the resolution 13C-NMR resonances, smce a sizable loss of the peak areas occurs when they contam immobthzed segments such as cross-links or crystallme porttons Figure 6 shows the proton decoupled “C-NMR and the ‘H-NMR spectra of PP-g-PDMAA membranes measured m DzO/H20. Only the resonance from the PDMAA chains (Huang et al, 1983) was clearly observed The explanation for this phenomenon is that these signals are due to the rapid motion of the grafted PDMAA chains swelling m D,O/H,O From these data, we concluded that the decrease of the water flux was caused mainly by the swelling of the grafted PDMAA chains Platelets adhesron test

iP-g-‘PDMAA

Fig. 4. Scanning electron nucrographs of ongmal PP membrane and PP-g-PDMAA membrane (%G = 12 1)

The number and shape change of platelets adhering on the polymeric surfaces were investigated with SEM, and the results are shown m Fig 7 A nonporous PP film was used as a substrate As we expected, DMAA-graftmg of the films’ surface effectively suppressed platelet adhesion and deformation, and only a small amount of type I platelets that is round m shape similar to native form, was observed (PgA-P and PgA-R) By contrast, a great number of adhered and spread platelets, type II N type III, were observed on the surfaces of PP and poly(methylmethacrylate). PDMAA IS a water-soluble polymer, so a PDMAA-grafted surface would become extremely hydrophilic and would swell when immersed m

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aqueous solutions. It has been reported that such hydrated surfaces, which have a very low interfacial free energy, decrease the adsorption and the denaturation of proteins, cells, and other homolecules (Ratner and Hoffman, 1980; Coleman et al., 1982; Ilcada et al., 1984). The vapor-phase LTP-graft techmque is very quick and easy to perform to obtain a highly hydrated surface compared to conventional grafting methods. Evaluation for plasma separation membrane The characteristics of the test membranes are listed m Table 1. Four membranes (PgA 1 - PgA4) of different %G, ranging from 12.1 to 18.9%, were prepared The surface PDMAA content, calculated by the data from ESCA and ATR-IR, increased with increasmg %G. This means that the surface IS not perfectly covered with PDMAA m a dry state. In

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other words, PDMAA and PP are mixed on the surface Relative plasma fluxes of PP-g-PDMAA membranes against those of non-grafted PP (100%) as shown in Fig. 8. PgA membranes, except PgA4, exhibited almost the same plasma filtration rate as the original PP membrane under a wall shear rate ranging from 150 to 500 s-i Though the water flux decreased, PgA Hrlth a lower grafting yield sulBctently filtered plasma from whole blood. It can be said that concentration polarization of red cells and platelets produced these results (Zydney et al., 1982) In suspenston filtration, like blood plasma separation, concentratton polanzatton dommates filtration kmetics As shown in Fig. 9, PgA membranes, except PgA4, showed sieving coefficients (SC) higher than 0.9 for albumm, total protein and total cholesterol. These data indicate that the PP-g-PDMAA membranes retam a sausfactory pore size as a plasma separator even in blood. As for PgA4, the amount of PDMAA grafted onto the surface of pore walls was too large to retam the nevmg coefficient and the plasma flux of the PP membrane Figure 10 shows the changes of SC for fibrinogen and F.VIII:C durmg plasma separation. Fibrinogen and F VIII:C are coagulant factors that are tmportant plasma proteins for transfusion. The SC of these proteins m the PP-g-PDMAA membrane was higher than that of non-grafted PP, especially in the early stage of filtration. We speculate that grafted PDMAA chains decreased the adsorption of the proteins onto the membrane surface. In contrast, PP surface adsorbs plasma proteins strongly due to hydrophobic interactions (Bras&in and Lyman, 1980).

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Membrane

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Thtckness (P)

Water flux (ml/mm m* mm Hg)

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130 130 130 130

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phylatoxms C3a and CSa are btoacttve polypepttdes that are produced durmg complement acttvatton, they can function as mediators of the acute mflam-

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matory responses, and serve as tmmunoregulatory factors Additionally, C5a can have direct effects on the pulmonary vasculature to increase pulmonary artery pressures and dtmmtsh cardiac output (Cheung et al, 1986) The chmcal srgmficance of C3a and C5a mediated immune response 1s still a matter of speculatton, however, the actrvatton of complement should be suppressed as much as posstble for donor-plasmapherests, because donor-plasmapherests IS employed to collect plasma from healthy donors. They don’t

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Complement actlvahon 1s an Important problem currently under dtscusslon (Ikeda et al, 1986, Chenoweth, 1988; Omokawa et al, 1989) The ana-

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Frg 11 The concentratrons of anaphylatoxms (C3a. C4a and C5a) m filtrated blood and outlet plasma* (0) filtrated plasma, (w) outlet blood obtain any medical benefit and hence the procedure must rnvolve essentially no nsk beyond that whrch a blood donor would encounter The concentratron of C3a, C4a and CSa anaphylatoxms m filtrated plasma and outlet blood from the module are grven m Fig 11 PP-g-PDMAA and original PP membranes showed lower increases

Acknowledgements-The authors are grateful to Tatsuya FuJn and Akrra Yagrshrta for expert techmcal assistance and valuable comments on blood plasma separation experiments The pernnsston by Terumo Corporatron to pubhsh this work IS greatly apprectated

m anaphylatoxms compared to CA membrane, espectally m filtered plasma The initial step m complement acttvatron when blood IS m contact wrth a bromatenal, IS thought to be a covalent binding of the labile thioester group of C3 to nucleophthc groups on the matenal surface. For example, hydroxy-groups contammg hydrophrhc materials, such as cullulose denvatrves and polyvmylalcohol, strongly activate complement (Low, 1983, Chenoweth, 1984). Hydromaterials such as polyethylene and phobic polypropylene, on the other hand, have a lower complement actlvatmg potential (Okamoto, 1987; Matsuda, 1987) However, hydrophobic surfaces adsorb plasma proteins strongly, and these proteins may serve as sites for complement activation. PDMAA-grafted surface is protein resistant, and relatrvely unreactive toward complement

Amett L M (1952) J Am Chem Sot. 74, 2027 Basxkm A and Lyman D J (1980) J Womed Mufer. Res. 14, 393 Chenoveth D E. (1984) Artlf. Organs 8, 281 Chenoveth D E (1988) Artlf. Organs 12, 502 Cheung A K, LeWmter M., Chenoweth D. E.. Lew W Y W and Henderson L. W. (1986) Kirlhey Jut. 29, 799 Clerk D T and Dilks A (1979) J. Pofym. Scr Po/ym Chem. Ed 17, 957 Clerk D T (1978) Polymer Surfaces (EdItedby Feast W. J.). Wiley, New York Coleman D L., Gregoms D. E., Andrade J. D. (1982). J. Blotned Mater. i&s 16, 381. Corbm G, A.. Cohen R E and Baddour R F 119851 Macromole&les 18, 98. Fales J. D., Bradley A and Howe R. F. (1976) Vactatm Technol (March), 53 Grasne N and Scott G (1985) Polymer Degradation and Stabrfrzatron. Cambridge University Press. Hollahan J. R. and Bell A T (Eds) (1974) Techniques and Appbcatlons of Plasma Cbemutry, Wiley, New York. Huang S. S. and Mcgrath J. E. (1983) Polym. Prepr. (ACS) 24, 138. Ikada Y ,Suxuki M. and Tamada Y. (1984) In Polymers as Biomaterrals (Ed&d by Shalaby S W., Hoffman A. S., Ratner B. D. and Horbett A. T ), p. 135. Plenum press, New York. Ikeda H , Hayammu H. and Tomono T (1986) Jpn J. Artrf. Organs 15, 1869. Jalfrin M J. (1989) J Membrane. Sci. 44, 115 Low S. (1983) Bio&em. J. 211, 381. Masuoka T.. Huasa 0.. Suds Y. and Onishi M. (1986) Proc. Conf on htatton &rtng Asta, Tokyo, p. 377. . Masuoka T., Huasa O., Suda Y and Omshr M. (1989) Radtat.Phys Cbem. 33,421.

CONCLUSIONS

A hydrophilic composite membrane, PP-gPDMAA, with an extremely hydrated surface, has been successfully obtained by LTP-graft of DMAA onto microporous PP. This membrane exhibited good separation capability of blood plasma, low complement activating potential and high blood compatibility. The vapor-phase LTP-graft is a quick and useful technique to prepare the surface of biomaterials, as well as to modify the interior surface of porous membranes.

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