Covalent Attachment of Phospholipid Analogous Polymers To Modify

Qian Yang, Zhi-Kang Xu, Zheng-Wei Dai, Jian-Li Wang, and Mathias Ulbricht ... Xiao-Jun Huang, Zhi-Kang Xu, Ling-Shu Wan, Zhen-Gang Wang, and Jian-Li ...
0 downloads 0 Views 491KB Size
Download PDF
Langmuir 2004, 20, 1481-1488

1481

Covalent Attachment of Phospholipid Analogous Polymers To Modify a Polymeric Membrane Surface: A Novel Approach Zhi-Kang Xu,*,† Qing-Wen Dai,† Jian Wu,*,‡ Xiao-Jun Huang,† and Qian Yang† Institute of Polymer Science and Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China Received October 15, 2003. In Final Form: December 1, 2003 A novel method for the surface modification of a microporous polypropylene membrane by tethering phospholipid analogous polymers (PAPs) is given, which includes the photoinduced graft polymerization of N,N-dimethylaminoethyl methacrylate (DMAEMA) and the ring-opening reaction of grafted poly(DMAEMA) with 2-alkyloxy-2-oxo-1,3,2-dioxaphospholanes. Five 2-alkyloxy-2-oxo-1,3,2-dioxaphospholanes, containing octyloxy, dodecyloxy, tetradecyloxy, hexadecyloxy, and octadecyloxy groups in the molecular structure, were used to fabricate the PAP-modified polypropylene membranes. The attenuated total reflectance FT-IR spectra of the original, poly(DMAEMA)-grafted, and PAP-modified membranes confirmed the chemical changes on the membrane surface. Scanning electron microscope pictures showed that, compared with the original membrane, the surface porosities of poly(DMAEMA)-grafted and PAP-modified membranes were somewhat reduced. Water contact angles measured by the sessile drop method on PAPmodified membranes were slightly lower than that on the original polypropylene membrane, but higher than those on poly(DMAEMA)-grafted membranes with the exception of octyloxy-containing PAP-modified membranes. However, BSA adsorption experiments indicated that the five PAP-modified membranes had a much better protein-resistant property than the original polypropylene membrane and the poly(DMAEMA)grafted membranes. For hexadecyloxy- and octadecyloxy-containing PAP-modified membranes, almost no protein adsorption was observed when the grafting degree was above 6 wt %. It was also found that the platelet adhesion was remarkably suppressed on the PAP-modified membranes. All these results demonstrate that the described approach is an effective way to improve the surface biocompatibility for polymeric membranes.

Introduction Polymer membranes have been widely used in water treatments, biochemical product separations, and biomedical applications. However, most polymer membranes used at present are fabricated from conventional materials such as polypropylene (PP), polyethylene (PE), poly(vinylidene difluoride) (PVDF), cellulose acetate (CA), polysulfone (PSf), poly(ether sulfone) (PES), poly(vinyl chloride) (PVC), and so on. When these membranes come into contact with protein or living organisms, the buildup of foulant on membrane surfaces and within pores will result in performance decline and product loss.1-2 Especially when polymer membranes are used in biomedical applications such as hemodialysis, the adsorption of proteins usually stimulates the attachment of fibrous and antibiotic moieties, leading to subsequent biological responses such as thrombus formation and immunoresponses.3 The extent of protein adsorption is mainly determined by the delicate balance of the interaction between the protein molecules and the solid substrate.3-8 * To whom correspondence should be addressed. Fax: ++86 571 8795 1773 (Z.-K.X.). E-mail: [email protected] (Z.-K.X.). † Institute of Polymer Science. ‡ Department of Chemistry. (1) Zeman, L. J.; Zydney, A. Microfiltration and Ultrafiltration: Principles and Apolypropylenelications; Marcel Dekker: New York, 1996. (2) Lloyd, D. R., Ed. Material Science of Synthetic Membrane; ACS Symposium Series 269; American Chemical Society: Washington, DC, 1985. (3) Horbett, T. A.; Brash, J. L. Protein at Interfaces II; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995. (4) Tsuruta, T., Hayashi, T., Kataoka. K., Ishihara, K., Kimura, Y., Eds. Biomedical Apolypropylenelications of Polymeric Materials; CRC Press: Boca Raton, FL, 1993. (5) Klee, D.; Ho¨cker, H. Adv. Polym. Sci. 2000, 149, 1.

Even if the influence of proteins is not counted, the nature of the substrate surface, including its hydrophilicity, charge type, and charge density, also has a strong influence on the structure and formation of the protein layer. Therefore, in recent years, there has been much interest in developing surface treatments to alter the chemical and physical properties of the substrate surface.4-8 It has been known for many years that hydrophilic polymer surfaces can reduce protein adsorption to some extent. Thus, the usual method of preparing biomaterials is to fabricate a hydrophilic layer onto the substrate surface. However, even on a hydrophilic surface, where the interaction of proteins with the material surface is entropically driven, the adsorption may also be irreversible because the accumulated number of direct contacts between protein fragments and the surface may be too large to allow desorption.3 Various polymeric materials offering improved biocompatibility have recently been developed.6-8 Among them, those containing phospholipid analogues are extremely effective to reduce protein adsorption, platelet deposition, and cell adhesion in vitro.9-11 The idea has evolved from the mimicry of cell membranes. It is well-known that the cell membrane is mainly composed of various phospholipid molecules. Proteins in the blood stream do not adsorb irreversibly onto the surface of cells, suggesting that their outer surface is truly biocompatible. At the early stage, it was thought that such biocompatibility was provided (6) Dillow, A. K., Lowman, A. M., Eds. Biomimetic Materials and Design; Marcel Dekker: New York, 2002. (7) Kasemo, B. Surf. Sci. 2002, 500, 659. (8) Shtilman, M. I. Polymeric Biomaterials. Part I. Polymer Implants; VSP: Zeist, The Netherlands, 2003. (9) Lewis, A. L. Colloids Surf., B 2000, 18, 261. (10) Ishihara, K. Sci. Technol. Adv. Mater. 2000, 1, 131. (11) Nakaya, T.; Li, Y. J. Prog. Polym. Sci. 1999, 24, 143.

10.1021/la035930l CCC: $27.50 © 2004 American Chemical Society Published on Web 01/13/2004

1482

Langmuir, Vol. 20, No. 4, 2004

Xu et al.

Figure 1. Schematic representative for the fabrication of PAP-modified membranes.

by the whole phospholipid molecule in the extracellular side of cell membranes. A great deal of effort was thereby made to immobilize phospholipid molecules onto the polymer surface to reproduce the biocompatibility effect.12-15 It was later suggested by Hayward and coworkers16-18 that the phosphorylcholine headgroup, instead of the whole molecule of phosphatidylcholine, is responsible for the blood compatibility. This improved understanding led to elevated activities to utilize phospholipid analogues in creating a biocompatible surface. Up to now, various vinyl-containing phospholipids and their polymer analogues were synthesized. Nakaya and Li11 summarized the phospholipid analogous polymers (PAPs) developed in the past 30 years. Meanwhile, the modifications of polymeric membranes with PAPs were also reported, which included coating,19-21 graft polymerization,22 in situ polymerization,14,15 and blending.23-24 However, the above methods all have their disadvantages; for example, the PAP blended with another polymer in a (12) Seitz, M.; Wong, J. Y.; Park, C. K.; Alcantar, N. A.; Israelachvili, J. Thin Solid Films 1998, 327, 767. (13) Weber, B. A.; Dodrer, N.; Regen, S. L. J. Am. Chem. Soc. 1987, 109, 4419. (14) Regen, S. L.; Kirszensztejn, P.; Singh, A. Macromolecules 1983, 16, 335. (15) Marra, K. G.; Winger, T. M.; Hanson, S. R.; Chaikof, E. L. Macromolecules 1997, 30, 6483. (16) Hayward, J. A.; Chapman, D. Biomaterials 1984, 5, 135. (17) Durrani, A. A.; Hayward, J. A.; Chapman, D. Biomaterials 1986, 7, 121. (18) Hayward, J. A.; Durrani, A. A.; Shelton, J.; Lee, D. C.; Chapman, D. Biomaterials 1986, 7, 126. (19) Akhtar, S.; Hawes, C.; Dudley, L.; Reed, I.; Stratfort, P. J. Membr. Sci. 1995, 107, 209. (20) Berrocal, M. J.; Johnson, R. D.; Badr, I. H. A.; Liu, M.; Gao, D.; Bachas, L. G. Anal. Chem. 2002, 74, 3644. (21) Iwasaki, Y.; Uchiyama, S.; Kurita, K.; Morimoto, N.; Nakabayashi, N. Biomaterials 2002, 23, 3421. (22) Ishihara, K.; Iwasaki, Y.; Ebihara, S.; Shindo, Y.; Nakabayashi, N. Colloids Surf., B 2000, 18, 325. (23) Hasegawa, T.; Iwasaki, Y.; Ishihara, K. Biomaterials 2001, 22, 243. (24) Uchiyama, T.; Watanabe, J.; Ishihara, K. J. Membr. Sci. 2002, 208, 39.

membrane matrix is easily run off during their operation for separation, and homopolymerization is inevitable during the graft polymerization of phospholipid analogues. Especially the preparation and purification of vinylcontaining phospholipids are troublesome, inefficient, nontime-saving, and cost-ineffective.9-10 In this work, we conceived an economic and efficient method to attach PAPs onto a polymer membrane, which is schematically described in Figure 1. The first step was the photoinduced graft polymerization of N,N-dimethylaminoethyl methacrylate (DMAEMA) onto a polypropylene membrane surface. Second, the poly(DMAEMA)grafted membrane was condensed with 2-alkyloxy-2-oxo1,3,2-dioxaphospholane to convert poly(DMAEMA) into a PAP. One advantage of this novel approach is that the graft polymerization of DMAEMA should be more efficient than those of vinyl-containing phospholipids because the large molecular size might cause a steric effect and diffusion difficulty for vinyl-containing phospholipids. Another advantage is that the purification step for the synthesis of vinyl-containing phospholipids could be avoided. Additionally, the alkyloxy groups in 2-alkyloxy2-oxo-1,3,2-dioxaphospholanes used for the reaction could be varied easily. In this study, the microporous polypropylene membrane was chosen as the substrate membrane for its high void volumes, well-controlled porosity, and low cost. To mimic the biomembrane, five 2-alkyloxy-2oxo-1,3,2-dioxaphospholanes, which had 8, 12, 14, 16, and 18 carbon atoms in the alkyloxy groups, were adopted. The photoinduced graft polymerization of DMAEMA, the fabrication of PAP-modified membranes, and the properties of the resultant membranes are described. Experimental Section Materials. A microporous polypropylene membrane with a porosity of 40% and an average pore diameter of 0.070 µm was prepared with the melt-extruded/cold-stretched (MECS) method in our laboratory. N,N-Dimethylaminoethyl methacrylate with 99% purity was favored by Xinyu Chemical Co. Ltd. (Wuxi, China), and was distilled under vacuum before use. Benzophe-

Attachment of PAPs To Modify a Polymeric Membrane none (BP), as an AR grade photoinitiator, was recrystallized three times before use. Bovine serum albumin (BSA; pI ) 4.8, Mw ) 66000) was purchased from Sino-American Biotechnology Co. Ltd. and used as received. Other reagents were AR grade and purified following normal procedures before use. 2-Chloro-2-oxo1,3,2-dioxaphospholane was synthesized according to the method of Lucus et al.25 and Edmundson,26 and the 102.5-104.5 °C/1 mmHg component was collected. Fresh human platelet-rich plasma (PRP) was bought from the Blood Center of Hangzhou, China. Synthesis and Analysis of 2-Alkyloxy-2-oxo-1,3,2-dioxaphospholanes. A thoroughly dried 500 mL three-necked flask was equipped with a mechanical stirrer, a drying tube, a themometer, and a dropping funnel. A 0.10 mol sample of dodecyl alcohol, 0.11 mol of triethylamine, and 150 mL dry tetrahydrofuran (THF) were added. When the solution was cooled to -10 to -5 °C, 0.1 mol of 2-chloro-2-oxo-1,3,2-dioxaphospholane was slowly added to the stirred solution over 2 h to keep the temperature constant. After the completion of addition, the reaction was continued at 10-15 °C with stirring for 2 h. The precipitated triethylamine hydrochloride was filtered off, and the filtrate was concentrated under reduced pressure to remove THF. This raw product, 2-dodecyl-2-oxo-1,3,2- dioxaphospholane, was purified by adsorption chromatography through a silica gel column with a mixture of acetone and chloroform as eluent. The chemical structure of the product was identified by FT-IR and 1H NMR spectra. (The FT-IR and 1H NMR spectra for 2-octadecyloxy-2-oxo-1,3,2-dioxaphospholane are shown in the Supporting Information.) The 1H NMR spectra were recorded at 500 MHz for solutions in CDCl3 at room temperature on a Bruck (Advance DM×500) nuclear magnetic resonance spectrometer, while the IR spectra of 2-alkyloxy-2-oxo-1,3,2-dioxaphospholane samples in KBr were obtained on a Bruck 22 FT-IR spectrometer. IR (KBr): 2924, 2853, 1446, 845, 722 cm-1, in which 722 cm-1 was due to the vibration of -(CH2)n-, n > 4, 1290 cm-1 was the absorbance band of the PdO vibration, and 1030 and 930 cm-1 corresponded to the vibrations of P-O-C. 1H NMR (CDCl3): δ ) 0.86-0.89 ppm (1t, 3H, -CH3), ∼1.25 ppm (1s, 30H, -CH2CH2(CH2)15CH3), 1.67-1.71 ppm (1m, 2H, -CH2CH2(CH2)15CH3), 4.12-4.16 ppm (1m, 2H, -CH2CH2(CH2)15CH3), 4.33-4.46 ppm (2m (adjacent), 4H, the four H atoms in the ring of 1,3,2dioxaphospholane). With a procedure similar to that for 2-dodecyloxy-2-oxo-1,3,2dioxaphospholane, 2-octyloxy-, 2-tetradecyloxy-, 2-hexadecyloxy-, and 2-octadecyloxy-2-oxo-1,3,2-dioxaphospholanes were also synthesized. There were no special differences in the IR and 1H NMR spectra between 2-octadecyloxy-2-oxo-1,3,2-dioxaphospholane and the other 2-alkyloxy-2-oxo-1,3,2-dioxaphospholanes, except that there were 10H, 18H, 22H, and 26H at δ ) ∼1.26 ppm in the 1H NMR spectra of 2-octyloxy-, 2-dodecyloxy-, 2-tetradecyloxy-, and 2-hexadecyloxy-2-oxo-1,3,2-dioxaphospholanes, respectively. Fabrication of Poly(DMAEMA)-Grafted Polypropylene Membranes. The graft polymerization of DMAEMA on the membrane surface was carried out using a 500 W ultra-highpressure mercury lamp at 25 °C. The wavelength was selected using a raster (350 ( 50 nm). The polypropylene membrane (thickness 20 µm, diameter 3.4 cm, area 9.08 cm2, mass 18.00 ( 2.00 mg) was coated with benzophenone as a sensitizer by the following procedure. The nascent microporous polypropylene membrane was washed with acetone three times to remove any chemicals adsorbed on the membrane surface, and dried in a vacuum oven at room temperature for 3 h before the initial weight was measured (W1). Then the membrane was immersed in benzophenone/acetone solution (3 g/dL) for 2 min. The membrane was pulled out and photoirradiated for 5 min in water. A 100 mL sample of DMAEMA/acetone solution was placed in a quartz tube, argon gas was passed through the solution for 5 min to eliminate any oxygen, and then the benzophenonecoated polypropylene membrane was moved into the DMAEMA/ acetone solution. The quartz tube was photoirradiated continuously for a definite time. After the graft polymerization, the (25) Lucas, H. J.; Mitchell, Jr. F. W.; Scully, C. N. J. Am. Chem. Soc. 1950, 72, 5491. (26) Edmundson, R. S. Chem. Ind. (London), 1962, October 20, 1828.

Langmuir, Vol. 20, No. 4, 2004 1483 membrane was picked out, washed with ethanol, acetone, and hot acetonitrile (60-70 °C), and then dried in a vacuum for 3 h. The weight of the grafted membrane was measured (W2). The grafting degree was calculated by

GDDMAEMA ) (W2 - W1)/W1 × 100

(1)

where W1 and W2 are the weights of the initial polypropylene membrane and the poly(DMAEMA)-grafted membrane, respectively. All results were the average of three parallel experiments at least. The standard error for the result was below 2% of the average value. Formation of PAPs on a Polypropylene Membrane. 2-Alkyloxy-2-oxo-1,3,2-dioxaphospholane, dried acetonitrile, and the poly(DMAEMA)-grafted polypropylene membrane were placed in a thoroughly dried 500 mL single-necked pressureresistant flask. Then, the flask was sealed to keep water out. Subsequently, the flask was placed in an oil bath at a temperature of 60-70 °C for the ring-opening reaction of 2-alkyloxy-2-oxo1,3,2-dioxaphospholane. During the reaction, the flask was shaken with a shaking speed of 120 rpm. After the reaction was completed, the membrane was taken out of the reaction flask and thoroughly washed with tetrahydrofuran. After drying, the membrane was weighed (W3). The reaction degree (RD) is defined as

RD ) {(W3 - W2)/Mw1}/{(W2 - W1)/Mw2} × 100

(2)

where W1, W2, and W3 are the weights of the virgin, poly(DMAEMA)-grafted, and PAP-modified membranes, respectively. Mw1 and Mw2 are the molecular weights of 2-alkyloxy-2-oxo-1,3,2dioxaphospholane and N,N-dimethylaminoethyl methacrylate, respectively. Characterization and Property Measurements. The attenuated total reflectance (ATR) FT-IR spectra of the original, poly(DMAEMA)-grafted, and PAP-modified polypropylene membranes were obtained on a Bruck 22 FT-IR spectrometer. The surface morphologies of the membranes were observed by scanning electron microscopy (SEM) using a Hitachi S-570 electron microscope. The static water contact angles of the membrane surface were measured by the sessile drop method and the captive bubble method at 25 °C with a contact angle goniometer (KRU ¨ SS DSA10-MK, Germany) equipped with video capture. Use of video capture for measuring the contact angle of porous materials had been discussed recently by Fisher et al.27 and Roudman et al.28 Following the reported processes, comparing these values between samples provided a semiquantitative measure of the differences in hydrophilicity for porous membranes and, to a certain extent, removed issues associated with porous media. In a typical acquisition for the sessile drop method, a water drop (∼50 µL) was dispensed on the membrane surface. Then, an image was recorded every 2 s, and a water contact angle was calculated from each image with the supporting software. The contact angle as a function of the drop age was plotted to determine a constant value. For the captive bubble method, the glass cell of the captive bubble assembly was filled with pure water. A membrane sample was then attached to a PMMA cover with double-sided tape. A specially designed and shaped syringe needle in the form of a “J” dispensed the air bubble from beneath the sample. The video capture provided an image of the captive bubble for the calculation of the contact angle. At least 10 contact angles were averaged to get a reliable value. BSA was used as a model protein to evaluate the proteinresistant characteristics of nascent and modified membranes. The studied membrane was immersed in ethanol for 10 min to prewet and then put into a BSA solution with various concentrations whose pH was adjusted to 8.0 with 0.1 M Tris-HCl buffer. This buffer can facilitate the hydrophobic interaction and inversely depress the electrostatic binding between the protein and the polymer surface.29 The mixture was incubated at 25 °C for 24 h to reach an adsorption-desorption equilibrium. The (27) Steen, M. L.; Jordan, A. C.; Fisher, E. R. J. Membr. Sci. 2002, 204, 341. (28) Roudman, A. R.; DiGiano, F. A. J. Membr. Sci. 2000, 175, 61.

1484

Langmuir, Vol. 20, No. 4, 2004

Figure 2. Effect of monomer concentration on the grafting degree of DMAEMA. amount of adsorbed protein was determined by measuring spectrophotometrically the difference between the concentrations of BSA in the solution before and after contact with the studied membranes. The spectroscopic analytical method utilized in this work for protein dosage was based on the reaction of albumin with Coomassie brilliant blue (Fluka) dyestuff to record the absorbance of the albumin-Coomassie brilliant blue complex according to Bradford’s method.30 A calibration curve between the spectrophotometrical absorbance and the BSA concentration was established to reduce the effect of protein adsorption at the surface of the experimental device for the adsorption measurements. The reported data are the mean values of triplicate samples for each membrane. A platelet adhesion experiment was carried out using the following procedure. First, the virginal, poly(DMAEMA)-grafted, and PAP-modified polypropylene membranes were treated with deionized water for 30 min at 50 °C and dried in a vacuum at room temperature. Fresh PRP which was obtained from 20 mL of human fresh blood by centrifugation at 1000 rpm for 10 min was used in all experiments. The studied membrane was cut into 1 × 1 cm pieces and placed on a tissue culture plate. A 20 µL sample of fresh PRP was dropped onto the center of the sample, which was then incubated at 37 °C for 30 min. The membrane was rinsed gently with a phosphate-buffered saline (PBS) solution, after which the adhered platelets were fixed with 2.5 wt % glutaraldehyde in PBS for 30 min. Finally, this sample was washed with PBS, and dehydrated with a series of ethanol/water mixtures of increasing ethanol concentration (30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% ethanol, 10 min in each mixture). The membrane surface was coated with gold and observed with SEM using a Cambridge S-260 scanning microscope.

Results and Discussion Photoinduced Graft Polymerization of DMAEMA. It is well-known that photoinduced graft polymerization is an effective and convenient method to modify the membrane surfaces.31-33 Here, this process was used to fabricate poly(DMAEMA)-grafted polypropylene membranes. Concerning the graft polymerization procedure of DMAEMA, the main factors influencing the grafting degree include monomer concentration and UV irradiation time. The effect of monomer concentration on the graft polymerization of DMAEMA is shown in Figure 2. It was found that the grafting degree increased gradually with an increase of the monomer concentration. Figure 3 shows (29) Xu, Z.-K.; Kou, R.-Q.; Liu, Z.-M.; Nie, F.-Q.; Xu, Y.-Y. Macromolecules 2003, 36, 2441. (30) Bradford, M. M. Anal. Biochem. 1976, 72, 248. (31) Ulbricht, M.; Richau, K.; Kamusewitz, H. Colloids Surf., A 1998, 138, 353. (32) Pieracci, J.; Crivello, J. V.; Belfort, G. J. Membr. Sci. 1999, 156, 223. (33) Ma, H.; Bowman, C. N.; Davis, R. H. J. Membr. Sci. 2000, 173, 191.

Xu et al.

Figure 3. Effect of UV irradiation time on the grafting degree of DMAEMA.

Figure 4. Effect of 2-alkyloxy-2-oxo-1,3,2-dioxaphospholane concentration on its ring-opening degree (reaction time 12 h, temperature 70 °C).

the effect of UV irradiation time on the grafting degree. It was also evident that the grafting degree of DMAEMA increased with an increase of the irradiation time. Therefore, a series of poly(DMAEMA)-grafted polypropylene membranes with different grafting degrees could be fabricated by tuning the monomer concentration and/ or UV irradiation time. Converting Poly(DMAEMA)-Grafted Membranes to PAP-Modified Membranes. It was reported in the literature34,35 that the ring-opening reaction of 2-alkyloxy2-oxo-1,3,2-dioxaphospholane was usually conducted at 60-80 °C. In our situation, 60 and 70 °C were selected because the pore structure of the polypropylene membrane might be damaged at relatively high temperature. The effects of 2-alkyloxy-2-oxo-1,3,2-dioxaphospholane concentration and reaction time on the ring-opening degree were studied. As can be seen from Figure 4, it seems that the ring-opening degree increased slightly with an increase of the 2-alkyloxy-2-oxo-1,3,2-dioxaphospholane concentration. This phenomenon might be ascribed to the adsorption of 2-alkyloxy-2-oxo-1,3,2-dioxaphospholane in the pores and on the surfaces of the membrane. Under the research conditions, the amount of 2-alkyloxy-2-oxo1,3,2-dioxaphospholane was more excessive than that of the grafted poly(DMAEMA); therefore, the reaction rate (34) Hofstee, B. H. J. Biochem. Biophys. Res. Commun. 1975, 63, 618. (35) Chen, T.-M.; Wang, Y.-F.; Li, Y.-J.; Kitamura, M.; Nakaya, T. Eur. Polym. J. 1997, 33, 273.

Attachment of PAPs To Modify a Polymeric Membrane

Langmuir, Vol. 20, No. 4, 2004 1485

Figure 5. Effect of reaction time on the ring-opening degree of 2-alkyloxy-2-oxo- 1,3,2-dioxaphospholane.

Figure 6. FT-IR/ATR spectra of the studied membranes: (a) original membrane; (b) poly(DMAEMA)-grafted membrane; (c) PAP-modified membrane.

was not determined by the concentration of 2-alkyloxy2-oxo-1,3,2-dioxaphospholane. Figure 5 shows the effect of reaction time on the ringopening degree at 60 and 70 °C. It was found that the reaction degree rapidly reached a high level (g95%) at 70 °C. It was obvious that increasing the temperature facilitated the ring-opening reaction. After 18 h of reaction, the reaction degree could reach 95% even at 60 °C. Characterization and Property Measurements. The poly(DMAEMA)-grafted and PAP-modified membranes were characterized by FT-IR/ATR spectroscopy to confirm the grafting of DMAEMA and the conversion of poly(DMAEMA) to a PAP. Figure 6 shows the FT-IR spectra of the original, poly(DMAEMA)-grafted, and PAPmodified membranes. Compared with Figure 6a, one vibration peak at 1730 cm-1 can be seen from the spectrum of the poly(DMAEMA)-grafted membrane (Figure 6b), which is attributed to the stretching vibration of CdO groups in poly(DMAEMA). As can be seen from Figure 6c, different from Figure 6b, there are new absorbance peaks at 1220, 1085, and 722 cm-1, which are attributed to the stretching vibrations of PdO, P-O, and the long alkyloxy group (-(CH2)n- (n > 4)), respectively. As mentioned in the Experimental Section, the stretching vibration of Pd O in the spectrum of 2-alkyloxy-2-oxo-1,3,2-dioxaphos-

Figure 7. SEM photographs of the studied membranes: (a) original membrane; (b) (b) 1.05 wt % poly(DMAEMA)-grafted membrane; (c) 1.25 wt % PAP-modified membrane; (e) 4.41 wt % poly(DMAEMA)-grafted membrane; (f) 4.65 wt % PAPmodified membrane.

pholane was at 1294 cm-1, but now it appears at 1220 cm-1 in the PAP-modified membrane because the ring of 2-alkyloxy-2-oxo-1,3,2-dioxaphospholane is opened. SEM was employed to examine the surface morphologies of typical modified membranes (Figure 7). Compared with the unmodified membrane, it was found that part of the surface porosity was somewhat plugged and some pores became smaller and more round in shape by the graft polymerization of DMAEMA, especially at higher grafting degree. On the other hand, compared with the poly(DMAEMA)-grafted membrane, it seems that the microporous structure of PAP-modified membranes was not obviously different. The hydrophilicity of the studied membranes was characterized on the basis of pure water contact angle measurements. It can be seen from Figure 8 that the water contact angle of poly(DMAEMA)-grafted membranes showed an obvious decrease when compared with that of the virginal membrane. However, after the ring-opening

1486

Langmuir, Vol. 20, No. 4, 2004

Xu et al.

Figure 8. Influence of grafting degree on the water contact angle of poly(DMAEMA)-grafted and PAP-modified membranes.

Figure 9. BSA adsorption on the poly(DMAEMA)-grafted and PAP-modified membranes.

reaction of poly(DMAEMA) with 2-alkyloxy-2-oxo-1,3,2dioxaphospholanes, the water contact angles of the corresponding PAP-modified membranes were higher than those of poly(DMAEMA)-grafted membranes except that octyloxy was the alkyloxy group in the PAP. The contact angles of octyloxy- containing PAP-modified membranes were much lower than those of poly(DMAEMA)-grafted membranes. This phenomenon might be explained by the molecular structure of the phospholipid analogous polymers. The long alkyloxy group of the PAP was hydrophobic. When the PAP-modified membranes were washed with THF and dried, the long alkyloxy groups fingered out, and a hydrophobic and successive upper surface was formed spontaneously on the polypropylene membrane surface. What contacted with water were not the moieties of the zwitterion but the moieties of the long alkyloxy group. It was also found from Figure 8 that no obvious differences were observed when the alkyloxy group in the PAP was longer than 14 carbons. However, the contact angle decreased with a reduction of the number of carbon atoms in the long alkyloxy group from 14 to 12, which was perhaps due to the imperfection of the long alkyloxy group surface. With the reduction of the number of carbon atoms in the long alkyloxy groups, the impact of the zwitterion moieties could reach the surface, which led to the decrease of the water contact angle. To prove this assumption, the contact angle of the PAPmodified membrane was measured by the captive bubble method also. The advantage of the captive bubble method over the sessile drop method was complete hydration of the sample, and thus, the surface energy of interest between water and the membrane should not change with the time of measurement. Typical results compared with the sessile drop method in an air atmosphere are listed in Table 1. It can be seen that, for PAP-modified membranes, the contact angle measured by the captive bubble method in water was much lower than the contact angle measured by the sessile drop method in air. On the other hand, the difference for a poly(DMAEMA)-grafted membrane could be omitted. This difference between the two measuring methods is a typical phenomenon caused by the conformation alteration of the hydrophilic macromolecular chains.36-38 It means that when the PAP-

Table 1. Water Contact Angles of the Poly(DMAEMA)-Grafted and PAP-Modified Membranes

(36) (a) Good, R. J. In Surface and Colloid Science; Good, R. J., Stromberg, R. R., Eds.; Plenum Press: New York, 1979; Vol. 11. (b) Andrade, J. D.; Smith, L. M.; Gregonis, D. E. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum Press: New York, 1985; Vol. 1, Chapter 7. (37) Yasuda, H.; Sharma, A. K.; Yasuda, T. J. Polym. Sci., Polym. Phys. Ed. 1981, 19, 1285.

contact angle (deg) sample no.

carbon no. in alkyloxy group

grafting degree (wt %)

sessile drop method

captive-bubble method

1 2 3 4 5 6

DMAEMA 8 12 14 16 18

6.0 5.7 6.2 6.6 6.1 6.1

85.7 ( 2.5 69.2 ( 2.3 93.3 ( 2.1 95.6 ( 2.3 96.2 ( 1.8 95.5 ( 1.9

81.7 ( 2.3 38.3 ( 2.1 40.1 ( 1.7 38.8 ( 1.6 38.9 ( 2.1 39.2 ( 1.5

modified membrane contacted with water for a long time, a turning-over occurred between the moieties of zwitterions and the moieties of long alkyloxy groups. Figure 9 shows the influences of grafting degree on BSA adsorption. It was found that the adsorbed BSA on the membrane decreased with an increase of the grafting degree for both poly(DMAEMA)-grafted and PAP-modified membranes at the studied range. The five PAP-modified membranes had better protein resistance than the unmodified polypropylene membrane and poly(DMAEMA)grafted membranes. Comparing the five PAP-modified membranes, nevertheless, with the increase of the number of carbon atoms in the alkyloxy groups from 14 to 16 and 18, BSA adsorption on the membrane surface was obviously suppressed. For hexadecyloxy- and octadecyloxycontaining PAP-modified membranes, almost no BSA adsorption was observed when the grafting degree was higher than 6 wt %. The reason for this was probably the mimetic characteristics of the PAP-modified membranes to a biomembrane. As we all know, the principal components of a biomembrane are lipids, proteins, and carbohydrates. However, the amount of proteins and carbohydrates is relatively small. Most of the lipids are phospholipids such as phosphatidylcholine, phosphadylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphatidylglycerol. These phospholipids all have two long alkyloxy groups in their molecules. Two layers of phospholipid molecules are facing each other, burying the hydrophobic moieties inside the membrane. The zwitterionic species cover the membrane surface. As mentioned previously by the contact angle measurements, there was a reorientation of the PAP to put the zwitterions at the surface of PAP-modified polypropylene membranes. During the protein adsorption measurements, the PAP-modified polypropylene mem(38) Tretinnikov, O. N.; Ikada, Y. Langmuir 1994, 10, 1606.

Attachment of PAPs To Modify a Polymeric Membrane

Langmuir, Vol. 20, No. 4, 2004 1487

Figure 10. Platelet adhesion on the original, poly(DMAEMA)-grafted, and PAP-modified membranes: (a) original membrane; (b) poly(DMAEMA)-grafted membrane with a grafting degree of 4.41 wt %; (c) dodecyloxy-containing PAP-modified membrane; (d) tetradecyloxy-containing PAP-modified membrane; (e) hexadecyloxy-containing PAP-modified membrane; (f) octadecyloxycontaining PAP-modified membrane.

branes were immersed in BSA solution for 24 h. In that case, the membranes were in an aqueous environment and should present the zwitterions at the surface. Thus, in these conditions, the membrane surfaces were hydrophilic and were relatively similar to that of the biomembrane. The hydrophilic surfaces normally facilitate reduction of protein adsorption on the membrane.1-2,27-29,31-33 Furthermore, with an increase of the number of carbon atoms of the long alkyloxy groups, the impact of the original membrane surface on protein adsorption could be reduced, which led to a decrease of protein adsorption on the PAPmodified membranes. Therefore, as we expected, the membranes showed an excellent property of protein resistance. Platelet adhesion was examined to evaluate the blood compatibility of the membrane surfaces. Figure 10 shows the SEM pictures for the original, poly(DMAEMA)-grafted, and PAP-modified membranes exposed to PRP for 30 min.

It was found that platelet adhesion on the membrane was strongly dependent on the chemical structure of the membrane surface. Numerous platelets adhered on the polypropylene membrane (Figure 10a), and some of them aggregated and deformed. Platelet adhesion on the poly(DMAEMA)-grafted membrane (Figure 10b) was much more serious than that on the original polypropylene membrane; however, the platelets had less deformation and scatter. Quite different from the original and poly(DMAEMA)-grafted membranes, platelet adhesion was effectively suppressed on the PAP-modified membranes. It can be seen from Figure 10c,d that platelet adhesion was hardly observed on PAP-modified membranes. The effect of grafting degree was studied with a sample of hexadecyloxy-containing PAP-modified membrane. The SEM pictures are shown in Figure 11. Compared with the original polypropylene membrane (Figure 10a), no obvious

1488

Langmuir, Vol. 20, No. 4, 2004

Xu et al.

Figure 11. Platelet adhesion on the hexadecyloxy-containing PAP-modified membranes with different grafting degrees. Grafting degree of PAP: (a) 0.58 wt %; (b) 3.89 wt %; (c) 5.69 wt %; (d) 6.88 wt %; (e) 8.13 wt %.

change was observed for platelet adhesion with an increase of the grafting degree of the PAP from 0.58 to 8.13 wt %. This result indicates that only 0.58 wt % PAP is enough to suppress the platelet adhesion on the polypropylene membrane. Conclusions PAPs can be conveniently attached onto a polymeric membrane by the photoinduced graft polymerization of DMAEMA followed by a ring-opening reaction with 2-alkyloxy-2-oxo-1,3,2-dioxaphospholanes. Both the conformation of the PAP and the carbon number in the long alkyloxy group had effects on the hydrophilicity of the membrane surface. With the exception of octyloxycontaining PAP-modified membranes, the water contact angles of the PAP-modified membranes were slightly lower than that of the original polypropylene membrane but higher than those of poly(DMAEMA)-grafted membranes. However, the PAP-modified membranes had a better

protein-resistant property than the unmodified polypropylene membrane and the poly(DMAEMA)-modified membranes. Furthermore, 0.58 wt % PAP was enough to suppress the platelet adhesion on the polypropylene membrane. As a conclusion, the surface biocompatibility of polymeric membranes could be improved effectively by tethering PAPs onto the membrane surface with the reported process. Acknowledgment. Financial support from the National Natural Science Foundation of China (Grant No. 20074033) and the High-Tech Research and Development Program of China (Grant No. 2002AA601230) is gratefully acknowledged. Supporting Information Available: FT-IR and 1H NMR spectra of 2-octadecyloxy-2-oxo-1,3,2-dioxaphospholane. This material is available free of charge via the Internet at http://pubs.acs.org.

LA035930L