Proteins at Interfaces II - American Chemical Society

Chapter 27

Reduced Protein Adsorption on Polymer Surface Covered with a Self-Assembled Biomimetic Membrane Kazuhiko Ishihara and Nobuo Nakabayashi

Downloaded by UNIV LAVAL on April 24, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch027

Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan

We have synthesized phospholipid polymers containing 2methacryloyloxyethyl phosphorylcholine(MPC) moieties as new blood compatible polymers and have evaluated their interactions with blood components. It was found that in the absence of anticoagulants blood clotting was delayed, and blood cell adhesion and activation were effectively prevented on the MPC copolymer surface. Protein adsorption on the MPC copolymer from human plasma was also reduced with increasing MPC fraction. The MPC copolymers are able to collect phospholipid molecules from plasma due to MPC moiety -phospholipid interactions and the accumulated phospholipids appear to create a self-assembled biomimetic membrane structure on the MPC copolymers which minimizes protein adsorption. When the MPC copolymer was treated with dipalmitoylphosphatidylcholine(DPPC) liposomal solution, DPPC molecules having the organized liposomal structure were adsorbed on the MPC copolymer surface. On the other hand, DPPC molecules were randomly adsorbed on conventional polymers. The amounts of plasma proteins adsorbed on the polymer surfaces decreased on pretreatment with DPPC liposomal solution for all polymers case, but the smallest amounts of adsorbed proteins were found on the MPC copolymer. The differences in protein adsorption on the different polymers probably reflect the differences in the state of organization of adsorbed phospholipid molecules on the polymer surfaces. In recent years, many polymer materials have been used for the fabrication of biomedical devices that contact blood, body fluids and tissues(7). Upon blood contact, biocomponents in blood such as lipids and proteins interact with these surfaces and are adsorbed on them. Well designed polymers are required to regulate their interactions. The adsorption state of these biocomponents strongly affects blood cell adhesion and activation which induce thrombus formation. The effect of proteins adsorbed onto polymer surfaces on nonthrombogenicity has been investigated by many researchers (2). Lyman et al. and K i m et al. claimed the importance of albumin adsorption for the nonthrombogenicity of a polymer surface (3,4). They proposed that predominant adsorption of albumin

0097-6156/95/0602-0385$12.00/0 © 1995 American Chemical Society Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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with a high adsorption rate and large equilibrium amount will make a polymer surface nonthrombogenic. K i m et al. also proposed that a polymer surface which can adsorb protein reversibly and/or minimally (or possibly no protein at all), will show minimal thrombogenicity (4). To minimize the amount of adsorbed protein, hydrophilic polymers, especially hydrogels have been prepared (5). The general results concerning protein adsorption on hydrogels clearly indicate that the amount and degree of denaturation of adsorbed protein decreases with increase in the water content of the hydrogel. Anderson et al. reported the adsorption and conformational change of proteins on hydrogels (6). We have also found that proteins are adsorbed from plasma to hydrogels such as acrylamide or vinylpyrrolidone copolymers even when their fractional water contents are higher than 0.4 (7). Moreover, some experimental results strongly suggested serious problems such as calcification, platelet deposition and complement activation when blood comes in contact with hydrogel surfaces (8). We have proposed the preparation of new nonthrombogenic polymers based on a different approach (9-18). We have noted that biomembrane structures which consist mainly of phospholipids and proteins show good biocompatibility since such surfaces interact only minimally with biocomponents. A similar approach was reported by Chapman etal.(19). They found that polymeric lipids and polyesters having a phosphorylcholine group are relatively nonthrombogenic based on thrombelastographic studies. It has also been reported that polyamide microcapsules coated with a lipid membrane reduced the adhesion of platelets(20). These studies indicated that the surface adsorbed with phospholipids interacted only minimally with blood cells. However, the effects of the lipid adsorption state on biocompatibility were not investigated. We synthesized copolymers having a phospholipid polar group, the 2-methacryloyloxyethyl phosphorylcholine(MPC) moiety, as a new type of biomaterial (11,15). We hypothesized that i f a biomembrane-like structure can be constructed on a polymer surface by spontaneously adsorbing natural lipids from a living organism, it will prevent the adsorption and activation of proteins and cells (14,16). It has already been reported that the M P C copolymers have an affinity for phospholipids (21,22) which were adsorbed on the surface in significant amounts (16,17). Moreover, the copolymers effectively suppressed thrombus formation (12,16,18). It was also found that the amount of adsorbed protein decreased with an increase in M P C content of the copolymers (13,16,18). In this review, the physical and protein adsorption properties of the M P C copolymers will be discussed with attention given to the probable creation of a biomembrane-like structure by organization of phospholipid molecules accumulated on the M P C copolymer surface. Materials and Methods Materials. M P C was synthesized by a previously reported procedure(77). The structure of M P C is shown in Figure 1. Poly[MPC-co-n-butyl methacrylate(BMA)], poly[2-hydroxyethyl methacrylate(HEMA)], and poly(BMA) were prepared by a conventional radical polymerization technique using 2,2'-azobisisobutyronitrile as initiator. The M P C mole fraction in the copolymer was determined by phosphorus analysis. In this study, we used two poly(MPC-C0-BMA)s with 0.25 and 0.16 M P C mole fractions. Cast films of each polymer were prepared by a solvent evaporation method. Dipalmitoylphosphatidylcholine(DPPC), bovine serum albumin(BSA), and bovine serum y-globulin(BSG) were obtained from Sigma Chemical Co. St Louis, M O , USA and used without further purification. Acrylic beads (250 - 600 pm diameter) or slabs (9 x 50 x 1 mm) were used as substrates onto which the polymers were

Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

27. ISHIHARA & NAKABAYASHI

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Reduced Protein Adsorption

coated. Polymer coating on these substrates was carried out by a solvent evaporation technique using a 0.5 wt% polymer solution (72, 14). The MPC mole fraction at the surface on the poly(MPC-a?-BMA) membrane was determined using X-ray photoelectron spectrometry (XPS, Shimadzu ESCA-750).


Evaluation of Organization of Phospholipid Adsorbed on Polymers Treated with Phospholipid Liposome. DPPC liposomes were prepared by the sonication method (14,21,22). Polymer membranes were incubated in the DPPC liposomal solution at 45 °C for 10 min. After washing with distilled water, thermal analysis was carried out by differential scanning calorimetry(DSC) to determine the gel-liquid crystalline phase transition temperature(Tc) of the DPPC liposome. Furthermore, by using a quartz resonator with gold electrodes coated with the poly(MPC-a?-BMA), the phase transition phenomena of the DPPC liposome adsorbed on the surface were observed (23). Determination of Amount and Conformational Change in Proteins Adsorbed on Polymers. The BSA and BSG were dissolved in PBS at a concentration of 4.5 g/dL and 1.6 g/dL, respectively, that is, the same concentration as in plasma. Acrylic slabs coated with polymer were immersed in 100 mL of protein solution at 30 °C. To avoid denaturation of the proteins in PBS, the maximum adsorption time was 30 min. The slabs were rinsed with PBS for 1 min, and then immersed in PBS in a quartz cell for spectroscopy. The amount of protein adsorbed on the surface was determined by ultraviolet(UV) spectrometry as described previously (14). For pre-adsorption of phospholipids on the polymer surface, the polymercoated acrylic slabs were immersed in the DPPC liposomal solution(0.5wt%) and incubated at 45 °C for 10 min. The amount of protein adsorbed on the polymer surface pretreated with DPPC was also measured by U V spectroscopy (14). Circular dichroism(CD) spectroscopic measurements on proteins adsorbed on the polymer surface was carried out to evaluate conformational change. A quartz slab was used as a substrate for the polymer coating and the polymer-coated quartz slab was immersed in the BSA solution for 60 min at 30 °C. The mean residual ellipticity of adsorbed B S A was calculated based on the method proposed by Akaike et al. (24) . Protein Adsorption on MPC Copolymer Surface When polymer materials contact blood, adsorption of lipids and proteins precedes cell adhesion. Thus protein adsorption not only on the polymer surface but also on the lipid adsorbed surface need to be clarified. Figure 2 shows the adsorption of BSA and BSG on three polymer surfaces. It is seen that adsorption increases with time in all cases. Baszkin and Lyman reported the maximum amount of adsorbed proteins calculated for a monolayer in an end-on type orientation, that is 0.90 |ig/cnr for BSA and 1.85 |ig/cm for B S G (25) . On hydrophobic poly(BMA), the adsorption rate of BSA was high and the adsorbed amount at 30 min was about 16 times higher than that for an end-on monolayer. Suggesting that multi-layer adsorption occurred. Similar adsorption behavior was observed for poly (HEM A). Compared with these polymers, the amount of BSA adsorbed on poly(MPC-a?-BMA) was quite low. Adsorption of BSG was also suppressed on poly(MPC-a>-BMA) compared with poly(BMA) and poly(HEMA), and was lower than the calculated value for a monolayer. Concerned with protein adsorption on the MPC copolymers, Sugiyama et al. also reported that the amount of B S A adsorbed on poly(MPC-c0-alkyl methacrylate) microspheres was lower than that on polystyrene microspheres and decreased with an increase in the hydrophilicity of the comonomer used for the preparation of the microspheres(26). z



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CH3 I CH2 = C C=0 O" I I + OCH2CH20POCH2CH2N(CH3)3 O

Figure 1. Structure of 2-methacryloyloxyethyl phosphorylcholine (MPC).

1

10 BSG

o in

G 9 O

10 20 30 Adsorption time (min)

40

s a < 0

-i

*

10 20 30 Adsorption time (min) C

40

Figure 2. Adsorption of BSA and BSG onto polymer surfaces at 30 C. (O) Poly(BMA), (A) Poly(HEMA), O Poly(MPC-a?-BMA), MPC mole fraction; 0.25. Initial concentrations of BSA and BSG were 4.5 g/dL and 1.6 g/dL, respectively (Reproduced with permission ref. 14).





389

Figure 3 demonstrates the C D spectra of BSA in PBS and adsorbed on the polymer surfaces (27). The molar ellipticity of native BSA dissolved in PBS was negative between 210 nm and 230 nm. In the case of BSA adsorbed on poly(HEMA), this value was slightly higher. A more significant change in the spectrum was found in the case of BSA adsorbed on poly(BMA), that is, the molar ellipticity was close to zero in the observed range. On the other hand, BSA adsorbed on poly(MPC-c0B M A ) had the same molar ellipticity as that of BSA in PBS. The ellipticity reflects the secondary structure of the protein, so it appears that no significant conformational change of BSA occurred during adsorption on poly(MPC-c0-BMA). It is considered that ^-potential is an important variable in relation to protein adsorption. We found that the ^-potential of a poly(ethylene terephthalate)(PET) membrane was -40.5 mV (Ueda, T.; Ishihara, K.; Nakabayashi, N. J. Biomed. Mater. Res., in press.), and coating of poly(HEMA) on the PET membrane reduced this value to -15.7 mV. Moreover, a poly(MPC-a?-BMA)-coated PET membrane showed a potential of -0.4 mV. In general, hydrophobic polymer surfaces have a negative ^-potential, whereas the potential approaches zero with increasing hydrophilicity. However, the fractional equilibrium water contents of membranes of poly(HEMA) and the poly(MPC- co-BMA) were very close to 0.38 and 0.39 respectively. Park and coworkers suggested that when proteins are adsorbed on a polymer surface via hydrophobic interactions, an exchange of bound water between the protein and the surface must take place (28). Therefore, we considered that the amount of bound water may be the key parameter for understanding the exceptionally mild interaction between proteins and the M P C copolymer surface. When DSC measurements of the hydrated polymer membranes were carried out to evaluate water structure, the fraction of free water(not bound water) in the poly(MPC-co-BMA) membrane was 0.74 and it found to be significantly higher than that in conventional hydrogels such as poly(HEMA)(0.41) (K.Ishihara, Tokyo Medical and Dental University, unpublished data.). Organized Adsorption of D P P C on M P C Copolymer We have previously reported protein and phospholipid adsorption on a poly(MPCC0-BMA) surface from human plasma (16). The amount of proteins adsorbed on the poly(MPC-c0-BMA) membrane decreased, while adsorption of phospholipid increased, with increasing MPC mole fraction of the copolymer. This result strongly suggested that preferential adsorption of phospholipid relative to protein occurred when the poly(MPC-co-BMA) came in contact with plasma. Moreover, it was also considered that the poly(MPC-co-BMA) might have specific affinity for the phospholipid in plasma. Therefore, we tried to clarify the interactions between phospholipids and poly(MPC-co-BMA) using DPPC as a model phospholipid (14, 17, 21, 22). Figure 4 indicates the relation between the amount of DPPC adsorbed on the poly(MPC-c0-BMA) and MPC mole fraction in the cppolymer(77). The amount of adsorbed DPPC on poly(BMA) was 2.14 ug/cm , and was the same as on poly(HEMA) even though the hydrophilicity of these materials are quite different. However, the adsorbed amount of DPPC on poly(MPC-co-BMA) is seen to depend on the M P C mole fraction, that is, the adsorbed amount of DPPC increases with increasing MPC mole fraction. This same tendency was observed on poly[MPC-c0styrene(St)] (27). The hydrophilicity of the M P C polymers increases with an increase in the M P C mole fraction (77, 75). These results suggest that the hydrophilic nature of the substrate is not the main factor determining the adsorption behavior of phospholipids. It is well known that phospholipid molecules adopt an organized structure in aqueous medium, presumably reflecting specific interactions


390


8 4 ©

0

o - o o

^

/

^

^

-4-1 -8


-12 -16 -20 200 210 220 230 240 250 260 Wavelength (nm) Figure 3. CD spectra of BSA in PBS and adsorbed on the polymer surfaces after contact with BSA solution for 60 min. ( ) in PBS, adsorbed on (O) poly(BMA), (A) poly(HEMA), and ( • ) poly(MPC-a>-BMA). MPC mole fraction : 0.16. Initial concentration of BSA: 0.45 g/dL (Reproduced with permission with ref. 27).

3.6

*P0.05 vs Poly(BMA)

3.2 2.8

•e 2.4 8

*0

Poly(HEMA)

i

Poly(BMA) 2.0 0 0.1 0.2 0.3 0.4 1.6 MPC mole fraction of poIy(MPC-co-BMA) Figure 4. Relationship between the amount of DPPC adsorbed on the poly(MPCco-BMA) and the mole fraction of MPC of the copolymer. The initial concentration of DPPC in the liposomal solution was 1.0 wt % which was prepared by sonication method.





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between phospholipid molecules. The increase in the amount of adsorbed DPPC may be due to the affinity of the M P C moiety for the DPPC molecule based on the "self-assembling" properties of the phospholipids since the M P C moiety has the same polar group as DPPC. When phospholipid molecules associate in an aqueous medium due to their amphiphilic nature and form a so-called multi- or bi-layered membrane structure, a gel-liquid crystalline transition of the long alkyl chain in the molecule is observed (29). Therefore, the adsorption state of DPPC molecules on polymer surfaces can be examined using DSC. Figure 5 shows DSC curves of polymer membranes treated with the DPPC liposomal solution (14). The DSC curve of the DPPC liposomal solution shows an endothermic peak at 41.8°C, corresponding to the Tc of the DPPC liposomes (29). If the bilayer membrane structure is destroyed, the endothermic peak will disappear. In the case of poly(BMA) and poly(HEMA) which were treated with the DPPC liposomal solution, there was no endothermic peak in the range of 20 °C to 50 °C (14). On the other hand, an endothermic peak was observed at 42 °C in poly(MPC-co-BMA) treated with the DPPC liposomal solution. There were no DSC peaks in that temperature range for any of the "conventional" polymer membranes which did not have M P C units. This result clearly indicates that the multi- and bi-layer membrane structure, as in the DPPC liposomes, was maintained on poly(MPC-co-BMA) whereas it was destroyed an the poly(BMA) and poly(HEMA) surfaces. Thus, it is considered that M P C moieties in the poly(MPC-co-BMA) play an important role in maintaining the bilayer membrane structure of adsorbed DPPC molecules. Very recently, we determined the phase transitions of DPPC liposomes adsorbed on the poly(MPC-coB M A ) which was coated on the quartz crystal(QC) microbalance as function of frequency change of the QC (23). Similar results were found for another M P C copolymer system: an endothermic transition was seen after addition of poly(MPCblock-SX) to a DPPC liposomal solution. On the other hand, it disappeared with the addition of poly(St) latex (21). Protein Adsorption on Poly(MPC-co-BMA) Treated with D P P C Liposome We considered that the interaction between proteins and the phospholipid adsorption layer depends on the orientation of the lipid molecules. Figure 6 shows the amount of proteins adsorbed on polymer surfaces which were pretreated with DPPC liposome. By comparison with Figure 2, it can be seen that the treatment of a polymer surface with a DPPC liposomal solution reduced BSA adsorption in the poly(BMA) and poly(HEMA) cases. For the case of poly(MPC-co-BMA), though the effect of DPPC treatment on protein adsorption seems very small, the amount of proteins adsorbed was reduced by about one-half compared to the bare polymer surface(first few minutes). The same effect of DPPC treatment on BSG adsorption was observed. The surface of the polymers becomes hydrophilic by adsorption of DPPC even if adsorption is random and the hydrophobic interaction between proteins and the surface is weakened. Moreover, when the polar groups of DPPC are structured on the surface, other interactions such as hydrogen bonding and/or electrostatic interactions seem to decrease. As previously mentioned, poly(MPC-ctf-BMA) appears to organize DPPC molecules adsorbed on the surface. Therefore, it is considered that the difference in protein adsorption between poly(MPC-co-BMA) and the other two polymers treated with DPPC is due to the difference in the organized state of DPPC molecules adsorbed on the surface. In conclusion, M P C copolymers adsorbed very little protein from plasma, possible due to the preferential adsorption of phospholipid molecules and the formation of a biomembrane-like organized adsorption layer of the phospholipids. M P C copolymers


392


I

DPPC liposomal solution Tc = 41.8 °C

DPPC liposome adsorbed on poly(BMA)


poly(HEMA)

D

poly(MPC-co-BMA)

37

39

41

45

43

Temperature (°C)

Figure 5. DSC curves of polymer membranes treated with DPPC liposomal solution. A: DPPC liposomal solution(2 wt%), B: poly(BMA), C: poly(HEMA), D: poly(MPC-co-BMA). MPC mole fraction, 0.25 (Reproduced with permission from ref. 14).

%

0

10

20

30

Adsorption time (min)

40

Proteins at Interfaces II - American Chemical Society

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