Reactions of Thrombresistant Multilayered Thin Films on Poly(vinyl

Quantitative ATR FT-IR analysis reveals that, when thin films are deposited by dipping, HP is immobilized to form covalent −CH2NH− linkages betwee...
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Langmuir 1998, 14, 7235-7244

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Reactions of Thrombresistant Multilayered Thin Films on Poly(vinyl chloride) (PVC) Surfaces: A Spectroscopic Study H. Kim and M. W. Urban* Department of Polymers and Coatings, North Dakota State University, Fargo, North Dakota 58105 Received January 5, 1998. In Final Form: September 28, 1998 These studies focus on the formation of multilayered thrombresistant thin films containing poly(ethylenimine) (PEI), dextran sulfate (DS), and heparin (HP), deposited on poly(vinyl chloride) surfaces. Dipping and spin-coating are employed to deposit multilayers, and using attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy, the effects of pH, concentration of surface reacting species, and cross-linking conditions on the formation of multilayered structures are investigated. Quantitative ATR FT-IR analysis reveals that, when thin films are deposited by dipping, HP is immobilized to form covalent -CH2NH- linkages between PEI-NH2, and terminal HP-CHO groups. However, when spin-coating is utilized as an application method, HP takes a parallel orientation, and ionic complexes between PEI-NH3+(OH-) and HP-SO3-(H3O+) are formed. Quantitative analysis indicates that the surface concentration of individual layers is significantly lower for spin-coated layers.

Introduction During the last decade, there has been considerable interest in the development of biomedical materials that would exhibit thrombresistant surfaces.1-3 For that reason, immobilization of heparin (HP) molecules on artificial surfaces has been extensively explored as one of the possible means to create thrombresistant surfaces,4-6 and nitrous acid degraded HP was often employed to generate these properties. However, these studies also indicated that in order to attach HP to polymeric surfaces, it is necessary to form several layers that will serve as a buffer between the blood stream and the surface of an artificial organ and create bonding sites available for surface reactions.7 This issue is particularly important when thermoplastic polymers, such as poly(vinyl chloride) (PVC), are used for artificial organs. Although several attempts to directly bond HP molecules to the PVC surface were made,7 relatively little success was met because HP molecules were physisorbed, instead of being covalently or ionically attached to the surface to provide sufficient thrombresistant activity. Therefore, in an effort to increase surface charge density and the content of functional groups for HP immobilization, multilayered structures were proposed. However, the surface and interfacial chemical structures responsible for thrombresistant activity of HP is unknown.4,8,9 The objective of this study is to elucidate the molecular origin of structures that develop in multilayered films * To whom all correspondence should be addressed. (1) Casu, B. Adv. Carbohydr. Chem. 1985, 43, 51. (2) Hoffman, J.; Larm, O. P.; Scholander, E. Carbohydr. Res. 1983, 117, 328. (3) Marcum, J. S.; Rosenberg, R. D. Biochem. Biophys. Res. Commun. 1985, 126, 365. (4) Larm, O. P.; Larsson, R.; Olsson, P. Biomat., Med. Dev., Art. Org. 1983, 11, 161. (5) Larm, O. P.; Adolfsson, L. A.; Olson, K. P. U.S. Patent 5,049,403, 1991. (6) Larmn, O. P. U.S. Patent 4,613,665, 1986. (7) Golander, C.; Larsson, R. U.S. Patent 4,565,740, 1986. (8) Fink, D. J.; Gendreau, R. M. Anal. Biochem. 1984, 139, 140. (9) Gendreau, R. M.; Leininger, R. I.; Winters, S. Adv. Chem. Ser. 1982, 199, 371.

that consist of poly(ethylenimine) (PEI), dextran sulfate (DS), and heparin (HP) attached to PVC surfaces. While chemical structures of the species involved in surface reactions are shown in Figure 1A, Figure 1B illustrates multilayered thin films that were deposited by dipping and spin-coating. Although the choice of depositing thin films using dipping and spin-coating exhibits appealing practical features, it appears that both application methods may lead to different surface species. As shown in Figure 1B, the first step will involve activation of the PVC surface by chemically generating negatively charged SO3-(NH4+) groups through the sulfonation reactions, followed by the formation of alternating cross-linked PEI and DS layers. The top un-cross-linked PEI layer is used to immobilize HP molecules, which form the outermost surface. Using attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy, these studies will investigate the effect of pH of the solution on the formation of thin films, the effect of concentration of the reacting species, and the effect of cross-linking conditions on the formation of multilayered thin films, followed by identification of reaction mechanisms responsible for HP immobilization. Experimental Section Substrate Preparation. PVC (Aldrich Chemical Co.; MW ) 75 000) was used as a substrate for depositing multilayered thin films. PVC (10 w/w %) in tetrahydrofuran (THF) was cast on a poly(tetrafluoroethylene) (PTFE)-coated plate, followed by drying the PVC specimen at room temperature for 24 h. Such prepared PVC films (20 × 20 × 1 mm) were allowed to dry in a vacuum desiccator for an additional 24 h to remove residual low molecular weight species (THF and H2O). Surface Reactions. Multilayered thin films were deposited on the PVC surface using solution exposure (dipping) and spincoating techniques. While dipping involved an exposure of PVC to a desired solution for 10 min, spin-coating was accomplished using a Laurell WS-200-4NPP/RV spin-coater using various shear rates from 5.9 to 74.8 s-1. The SO3-(NH4+) groups were attached by dipping or spincoating aqueous solution of (NH4)2S2O8 (0.1-0.8 g/mL) at 80 °C, followed by reactions with H3BO3 (0.7-5.9 g/mL) in order to oxidize the surface.

10.1021/la980006q CCC: $15.00 © 1998 American Chemical Society Published on Web 11/04/1998

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Figure 1. (A) Chemical structures used in surface reactions. (B) Schematic representation of multilayered structures. An aqueous solution (5 w/w %) of PEI (H2N(CH2CH2NH)nH) (0.005-0.16 g/mL) was stabilized on the sulfonated PVC through cross-linking reactions with crotonaldehyde (CH3CHdCHCHO) at 80 °C. The pH of the solution was adjusted from 6 to 11 using 4 M NaOH and 2 M HCl, and the crotonaldehyde concentration was changed from 0.0008 to 0.0117 mol/L at pH 10. DS (H(OCH2C4H3(SO3-)(H3O+)(OH)2OCH)nH) was applied to the PEI-coated surface at 80 °C. The pH of the solution was varied from 2 to 7 using 4 M NaOH and 2 M HCl, and the DS concentration was changed from 0.005 to 0.16 g/mL. Thereafter, alternating layers of cross-linked PEI and DS, followed by a top layer of un-cross-linked PEI, were reacted at 80 °C. After each step of the reaction, the PVC film was washed with distilled water and dried in a vacuum deccicator for 24 h. HP (OCH2C4H4(OH)2O(SO3-)(H3O+)CHO) was immobilized on a cross-linked and an un-cross-linked PEI surface in the presence of 1.5 M NaCl and 0.39 M sodium cyanoborohydride (NaCNBH3) at 80 °C. The pH was controlled from 2 to 7 using 4 M NaOH and 2 M HCl, and the HP concentration was varied from 0.0025 to 0.12 g/mL. Spectroscopic Measurements. ATR FT-IR spectra were collected on a Nicolet Magna-IR single beam spectrometer. A resolution of 4 cm-1 and a mirror speed of 0.3165 cm s-1 were used. The ATR cell was aligned at a 60° angle of incidence using a 45° angle parallelogram KRS-5 crystal. In an effort to determine orientation of the surface species, 90° (TE) and 0° (TM) polarized infrared light was used. TE is a transverse electric vector of the incidence light polarized at 90° with respect to sample surface,

whereas TM is a transverse magnetic vector polarized at 0° with respect to sample surface. Each spectrum represents 200 coaded scans ratioed against a reference spectrum obtained from 200 coaded scans of an empty ATR cell. All spectra were corrected for spectral distortion using Q-ATR software.10,11 In order to determine the extinction coefficient of the hydroxyl groups, various concentration standards of PEI, DS, and NAD heparin were prepared, and transmission spectra were obtained. Transmission spectra were collected on a Nicolet Magna-IR single beam spectrometer at a resolution of 4 cm-1, and with a mirror speed 0.3165 cm s-1. Quantitative analysis was performed using Q-DEPTH software.

Results and Discussion As stated in the Introduction, the objective of these studies is to produce thrombresistant surfaces by attaching thin films of PEI, DS, and HP to the PVC surface. Two methods of depositing multilayered structures will be explored: solution exposure (dipping) and spincoating. Because of the distinctly different nature of both processes, they will be discussed in separate sections. (10) Urban, M. W. Attenuated Total Reflectance Spectroscopy of PolymerssTheory and Practice; American Chemical Society: Washington, DC, 1996 (see also references therein). (11) Urban, M. W. Vibrational Spectroscopy of Molecules and Macromolecules on Surfaces; Wiley-Intersciences: New York, 1993.

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Figure 2. ATR FT-IR spectra in the S-O stretching region for ammonium persulfate reacted with the PVC surfaces: (A) unreacted PVC; (B) ammonium persulfate at 0.1 g/mL; (C) ammonium persulfate at 0.2 g/mL; (D) ammonium persulfate at 0.4 g/mL; (E) ammonium persulfate at 0.8 g/mL.

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Figure 3. ATR FT-IR spectra in the N-H stretching region for PEI reacted with the PVC surfaces for dipping conditions: (A) cross-linked first layer of PEI at 0.005 g/mL; (B) crosslinked first layer of PEI at 0.01 g/mL; (C) cross-linked first layer of PEI at 0.02 g/mL; (D) cross-linked first layer of PEI at 0.04 g/mL; (E) cross-linked second layer of PEI at 0.04 g/mL; (F) un-cross-linked third layer of PEI at 0.04 g/mL.

Table 1. Assignment of Characteristic IR Bands functional groups sulfonated PVC -SO3-(NH4+) PEI -CH2CH2NH-CHdNDS -SO3-(H3O+) HP -SO3-(H3O+) -NHSO3-(H3O+) -CH2SO3-(H3O+)

IR band (cm-1)

assignment

1023 1254

SsO symmetric stretching SsO asymmetric stretching

1650 1560

NsH deformation CdN stretching

1642

OsH bending

1623 3358 3422

OsH bending OsH stretching OsH stretching

Multilayered Thin Films Obtained by Solution Exposure (Dipping). According to Figure 1B, the first layer can be attached by sulfonating the PVC surface with (NH4)2S2O8, followed by oxidizing it with H3BO3. Figure 2 illustrates ATR FT-IR spectra of the sulfonated PVC surface in the S-O stretching region. While trace A is the spectrum of the unreacted PVC surface, trace B represents the spectrum of the sulfonated PVC surface. It appears that the new band at 1023 cm-1 is detected. As shown in Table 1, this band is attributed to the S-O symmetric stretching modes,12 resulting from the formation of the SO3-(NH4+) groups on the PVC surface. On the other hand, an increase of the band at 1254 cm-1 attributed to the S-O asymmetric stretching mode12 of SO3-(NH4+) groups is detected. When the concentration of the (NH4)2S2O8 solution increases from 0.1 (trace B) to 0.8 g/mL (trace E), the intensities of the S-O stretching bands detected at 1023 cm-1 and 1254 cm-1 also increase. These observations indicate that the formation of the SO3-(NH4+) groups introduced on the PVC surface is a function of the (NH4)2S2O8 solution concentration. (12) Bellamy, L. J. The Infrared Spectra of Complex Molecules; John Wiley & Sons: New York, 1975.

The next layer to be reacted is PEI (Figure 1B), which will form a cross-linked layer. Figure 3 illustrates ATR FT-IR spectra in the NsH deformation region of PEI reacted to the PVC surface. Trace A illustrates the spectrum of the first layer of PEI cross-linked to the previously sulfonated PVC surface. It appears that new bands are detected at 1650 and 1560 cm-1 and attributed to the NsH deformation modes of PEI and CdN stretching modes of cross-linked PEI,13 respectively. As the PEI solution concentration increases from 0.005 (trace A) to 0.04 g/mL (trace D), the intensities of both bands increase, indicating that PEI is attached to the sulfonated PVC surfaces and cross-linked through the CdC opening of crotonaldehyde to form CdN bonds. As shown in Figure 1B, after the DS layer is reacted, the second layer of PEI is cross-linked to the DS surface. Trace E represents the spectrum of PEI cross-linked to the DS surface. When 0.04 g/mL of PEI solution concentration is employed, the intensities of both bands detected at 1650 and 1560 cm-1 increase, as compared to the PEI cross-linked to the previously sulfonated PVC surface, indicating that DS molecules provide a good source for increasing the effective concentration of the SO3-(H3O+) groups to react with PEI. After two alternating layers of cross-linked PEI and DS are deposited on the sulfonated PVC surface, the top layer of un-cross-linked PEI is added in order to increase the amount of terminal NH2 groups on the surface. Trace F represents the spectrum of un-cross-linked PEI reacted to the DS surface. The intensity of the N-H deformation band detected at 1650 cm-1 decreases, and the CdN stretching band detected at 1560 cm-1 disappears due to a lack of the PEI cross-linking reactions. This is desirable because the primary reason for the formation of un-crosslinked PEI layers on the outermost surface is to provide the high content of NH2 species in order to enhance HP immobilization. (13) Barcello; Bellanato. Spectrochim. Acta 1956, 8, 27.

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Figure 4. ATR FT-IR spectra in the O-H bending region for DS reacted with the PVC surfaces for dipping conditions: (A) first layer of DS at 0.005 g/mL; (B) first layer of DS at 0.01 g/mL; (C) first layer of DS at 0.02 g/mL; (D) first layer of DS at 0.04 g/mL; (E) first layer of DS at 0.08 g/mL; (F) second layer of DS at 0.08 g/mL.

As shown in Figure 1B, the next layer attached to the sulfonated PVC surface and cross-linked PEI is DS. Figure 4 illustrates ATR FT-IR spectra in the O-H bending region for DS reacted to cross-linked PEI surfaces. The new band developed at 1642 cm-1 is attributed to the O-H bending modes of DS-SO3-(H3O+) groups.14 As the DS solution concentration increases from 0.005 (trace A) to 0.08 g/mL (trace E), the 1642 cm-1 band intensity also increases. These data indicate that DS is attached to the cross-linked PEI surface and the thickness of this layer depends on the DS solution concentration. As previously mentioned, the spectrum of the second PEI layer exhibits the higher intensity of the N-H deformation band. Therefore, DS is reacted to the second PEI layer in order to increase the SO3-(H3O+) concentration. Trace F represents the spectrum of DS reacted to the second layer of the PEI surface. A comparison of the spectra of the second layer with the first DS layer indicates that the band intensity at 1642 cm-1 due to O-H bending modes increases. The depth penetration of IR light reaches, 0.8 µm from the top surface into the DS layer. In this range, the 1650 cm-1 band due to the second layer of PEI was not detected. Therefore, trace F of Figure 4, the spectrum of the second layer of DS, is not affected by the first layer of DS. These observations indicates that the effective concentration of the SO3-(H3O+) groups of the second DS layer is increased, thus providing more reactive sites for PEI. The next step involves reactions of HP to the modified PVC surface. Figure 5 illustrates ATR FT-IR spectra in the O-H bending region for HP deposited on the previously modified PVC surface. A new band detected at 1623 cm-1 is attributed to the O-H bending modes of HP-SO3-(H3O+) groups. As the HP solution concentration increases from 0.0025 (trace A) to 0.12 g/mL (trace F), intensities of the bands at 1623 cm-1 also increase. As the HP solution concentration increases, another band develops at 1650 (14) Socrates, G. Infrared Characteristic Group Frequencies; WileyInterscience: New York, 1980.

Kim and Urban

Figure 5. ATR FT-IR spectra in the O-H bending region for HP reacted with the PVC surfaces for dipping conditions: (A) HP at 0.0025 g/mL; (B) HP at 0.005 g/mL; (C) HP at 0.01 g/mL; (D) HP at 0.03 g/mL; (E) HP at 0.06 g/mL; (F) HP at 0.12 g/mL.

cm-1, which is attributed to the N-H deformation modes.13 As previously mentioned, HP contains SO3-(H3O+) and terminal CHO groups. Thus, both groups can react with PEI: one reaction would involve HP ionic bonding through the formation of ionic complexes between PEI-NH3+(OH-) and HP-SO3-(H3O+) groups, and the other is covalent bonding through the formation of the -CH2NH- linkages between PEI-NH2 and HP-CHO groups. Therefore, the presence of the N-H deformation band detected at 1650 cm-1 indicates that HP becomes covalently bonded to the PEI-modified PVC surface through the formation of -CH2NH- linkages. On the other hand, the band detected at 1673 cm-1 is attributed to the N-H deformation mode of unreacted PEI-NH3+(OH-) species. For reference purposes, if the ionic complex was formed between PEI-NH3+(OH-) and HP-SO3-(H3O+) groups, one would anticipate the presence of the 1661 cm-1 band attributed to the N-H deformation mode of -NH3+SO3-- ionic complexes. The proposed reaction mechanisms responsible for HP immobilization are depicted in Figure 6. It should be kept in mind that the above data were obtained for specimens for which every layer was deposited by direct solution exposure (dipping). Quantitative Analysis. Although the objective of many surface studies is determination of a chemical makeup of a surface, another objective is to be able to quantify newly formed species. For this reason, intensities of the bands responsible for the above surface reactions were measured and used to quantify each reacted layer. While the CdN stretching at 1560 cm-1 and the NsH deformation at 1650 cm-1 bands will be used as a measure of the cross-linked and un-cross-linked PEI reactions, the O-H bending bands of DS-SO3-(H3O+) and HP-SO3-(H3O+) at 1642 and 1623 cm-1 will be used to determine the extent of DS and HP reactions on the PVC surface. Since quantitative infrared analysis requires knowledge of extinction coefficients of these bands,10 Table 2 lists extinction coefficients for 1650, 1560, 1642, and 1623 cm-1 bands obtained by measuring known concentrations of reactive species. Using these values, the Beer-Lambert law (β ) c, where  is the extinction coefficient, c is the

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Figure 6. Proposed reaction mechanism of HP immobilization through dipping. Table 2. Extinction Coefficients of the Characteristic Band for PEI, DS, and HP species

IR band (cm-1)

extinction coefficient  (cm2/g)

cross-linked PEI un-cross-linked PEI dextran sulfate heparin

1560 1650 1642 1623

621.12 907.98 623.54 1611.45

concentration, and β is the linear absorptivity) allows us to determine known concentrations in a transition mode of detection. However, in order to accomplish quantitative analysis, it is necessary to correct ATR spectra for optical effects10,11 and utilize proper quantitative algorithm. For that reason, the absorption index spectrum is refined by an iterative process that minimizes the difference between the true and calculated reflectivity resulting from optical effects.10,11 At the same time, the Kramers-Kronig relationship between absorption (k) and refractive (n) indices is maintained.10,11 This iterative process is used in conjunction with numerical double Kramers-Kronig transformation (KKT) method to obtain ATR spectra suitable for quantitative analysis. Using corrected ATR spectra, linear absorbtivity is obtained to allow calculations of volume concentrations from the Beer-Lambert law. Based on the volume concentrations, surface concentrations are obtained by multiplying the penetration depths of characteristic IR bands. Further details involved in the applications and use of this algorithm which accounts for distortions of strong and weak bands as well as quantitative ATR measurements can be found in the literature.10,11,15,16

Using the above methodology, Figure 7a was constructed, which illustrates the surface concentration changes of cross-linked PEI on sulfonated PVC as a function of pH. It appears that for the pH values ranging from 6 to 8, the surface concentration of PEI is 2.80 × 10-4 mg/m2 (3.01 × 102 mg/m3). As the pH changes from 8 to 10, the PEI concentration increases linearly from 2.80 × 10-4 (3.01 × 102) to 0.96 × 10-3 mg/m2 (1.03 × 103 mg/m3), to reach a steady state at 0.94 × 10-3 mg/m2 (1.01 × 103 mg/m3) when pH > 10. These observations indicate that higher PEI concentrations are obtained under basic conditions. Figure 7b illustrates surface concentration changes of cross-linked PEI on the sulfonated PVC surface plotted as a function of crotonaldehyde concentrations. As the solution concentration of crotonaldehyde changes from 0.08 × 10-2 to 0.48 × 10-2 mol/L, the cross-linked PEI surface concentration increases from 2.73 × 10-4 (2.94 × 102) to 1.41 × 10-3 mg/m2 (1.52 × 103 mg/m3). However, when higher concentration levels of crotonaldehyde above 0.48 × 10-2 mol/L are employed, PEI cross-links in the solution and precipitates out, thus preventing further surface reactions with PEI. For this reason, the crosslinked PEI surface concentration decreases above 0.48 × 10-2 mol/L of crotonaldehyde. Figure 7c illustrates surface concentrations of crosslinked and un-cross-linked PEI on the sulfonated PVC surface plotted as a function of PEI solution concentration. Curve A represents surface concentration changes of the (15) Huang, J. B.; Urban, M. W. Appl. Spectrosc. 1992, 46 (11), 1666. (16) Huang, J. B.; Urban, M. W. Appl. Spectrosc. 1993, 47 (7), 973.

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Figure 8. (a) Surface concentration of DS as a function of pH for dipping conditions. (b) Surface concentration of DS reacted to PVC surfaces as a function of concentration for dipping conditions: (A) first layer; (B) second layer.

Figure 7. (a) Surface concentration of PEI as a function of pH for dipping conditions. (b) Surface concentration of PEI as a function of cross-linker content for dipping conditions. (c) Surface concentration of PEI as a function of concentration for dipping conditions: (A) cross-linked first layer; (B) cross-linked second layer; (C) un-cross-linked third layer.

first cross-linked PEI layer on sulfonated PVC surface, such as shown in Figure 1B. As the PEI solution concentration changes from 0.005 to 0.04 g/mL, its surface concentration increases from 8.56 × 10-4 (9.20 × 102) to 1.47 × 10-3 mg/m2 (1.58 × 103 mg/m3). In the second layer, the surface concentration of PEI changes from 8.57 × 10-4 (9.21 × 102) to 1.65 × 10-3 mg/m2 (1.77 × 103 mg/ m3). This is shown by curve B of Figure 7c. As previously mentioned, the first layer of PEI is cross-linked to the sulfonated PVC surface, but the second layer of PEI is cross-linked to the DS surface. Therefore, the higher PEI concentration of the second layer is attributed to the fact that DS provides a higher content of the SO3-(H3O+) groups on the surface, thus providing more opportunities to react with PEI. When the PEI solution concentration is above 0.04 g/mL, surface concentrations of both layers remain unchanged. It should be noted that the formation of the third PEI-DS layer was accomplished using uncross-linked PEI in order to provide higher NH2 concen-

trations accessible for bonding with HP, thus enhancing immobilization of HP molecules. The surface concentration changes of PEI in the third layer were quantified by using the extinction coefficient obtained for the N-H deformation band at 1650 cm-1. As shown in Figure 7c, curve C, concentration levels range from 2.63 × 104 (3.02 × 102) to 4.89 × 10-4 mg/m2 (5.62 × 102 mg/m3), when the PEI concentration in the solution changes from 0.005 to 0.04 g/mL. Compared to crosslinked PEI layer, lower surface concentration was obtained because uncrosslinked PEI layer is water-soluble. With these results in mind, let us consider how the pH of the solution will affect surface reactions of DS. In order to form charged surface with NH3+(OH-) groups for further reactions, the surface should be treated with acidic solution. For that reason, acidic solution (pH < 6) was employed. Figure 8a illustrates surface concentration changes of the first DS layer on PEI plotted as a function of pH. As seen, the DS surface concentration is similar to the surface concentration of PEI, but when the pH changes from 2 to 4, the surface concentration of DS increases from 7.51 × 10-5 (8.53 × 10 mg/m3) to 1.77 × 10-4 mg/m2 (2.01 × 102 mg/m3). The pH changes from 4 to 6 result in a linear increase, from 1.77 × 10-4 (2.01 × 102) to 2.07 × 10-3 mg/m2 (2.35 × 103 mg/m3). Similarly to the PEI surface concentration, the DS surface concentration levels off at 2.24 × 10-3 mg/m2 (2.55 × 103 mg/m3), when pH > 6. These observations indicate that higher surface concentrations of DS can be obtained under mild acidic conditions (4 > pH > 6) because the PEI surface exhibits positive NH3+(OH-) groups under acidic conditions. On the other hand, when pH < 4, lower surface concentrations of DS are obtained because most of the DS-SO3-(H3O+) associations lose their ionic strength to form DS-SO3H associations. The effect of DS solution concentration on surface reactions is plotted in Figure 8b. Curve A illustrates the

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Figure 10. ATR FT-IR spectra in the O-H bending region for HP reacted with the PVC surface for spin-coating conditions: (A) HP at 0.005 g/mL, (B) HP at 0.01 g/mL; (C) HP at 0.03 g/mL; (D) HP at 0.05 g/mL; (E) HP at 0.10 g/mL. Figure 9. (a) Surface concentration of HP as a function of pH for dipping conditions. (b) Surface concentration of HP as a function of concentration for dipping conditions: (A) HP immobilized on the cross-linked PEI surface; (B) HP immobilized on the un-cross-linked PEI surface.

surface concentrations of the first layer and ranges from 1.24 × 10-3 (1.41 × 103) to 6.20 × 10-3 mg/m2 (7.05 × 103 mg/m3) when the DS solution concentration was changed from 0.005 to 0.08 g/mL, respectively. On the other hand, the concentration of the second layer increases from 1.28 × 10-3 (1.45 × 103) to 7.18 × 10-3 mg/m2 (8.16 × 103 mg/ m3). This is shown in curve B, Figure 8b. As previously mentioned, the second PEI layer exhibits higher surface concentration levels than the first PEI layer. Higher surface concentration levels of the second DS layer result from the increased surface concentration of the second PEI layer, which will provide more opportunities to react with DS-SO3-(H3O+) groups. On the other hand, surface concentrations of both DS layers level off above 0.08 g/mL, indicating that the PEI film no longer supplies positive NH3+(OH-) groups for further reactions with DS. As indicated earlier, the primary objective of these studies is to make the PVC surface thrombresistant by attaching HP to PVC. Figure 9a illustrates surface concentration changes of HP immobilized on the multilayer-modified PVC surface (Figure 1B) plotted as a function of pH. There are two reactive sites for HP that can potentially react with the previously created PEI surface: HP-CHO and HP-SO3-(H3O+) entities. Multilayered coatings produced by direct exposure to respective solutions show that HP is immobilized through the formation of the -CH2NH- linkages between HP-CHO and PEI-NH2 groups. This was demonstrated by the formation of the 1650 cm-1 band due to N-H deformation modes of -CH2NH- linkages, and the reactions responsible for the covalent bond formation are illustrated in Figure 6. When the pH changes from 7 to 5, the surface concentration of HP immobilized on the PEI surface remains constant. On the other hand, as the pH values decrease from 5 to 2, the surface concentration of HP increases from 1.10 × 10-4 (1.24 × 102) to 1.87 × 10-3

mg/m2 (2.10 × 103 mg/m3). These observations indicate that HP-CHO is activated when acidic conditions below pH 5 are utilized, resulting in immobilization on the outermost PEI layer. The effect of the HP solution concentration on surface reactions is plotted in Figure 9b. Curve A illustrates the surface concentration of HP immobilized on the crosslinked PEI surface, which ranges from 2.06 × 10-3 (2.31 × 103) to 3.10 × 10-3 mg/m2 (3.48 × 103 mg/m3), for the HP solution concentration changes ranging from 0.0025 to 0.12 g/mL. On the other hand, surface concentration of HP immobilized on the un-cross-linked PEI surface exhibits higher yields and increases from 6.63 × 10-3 (7.45 × 103 mg/m3) to 1.58 × 10-2 mg/m2 (1.78 × 104 mg/m3), compared to its concentration level on the cross-linked PEI surface. This is shown in Figure 9b, curve B and indicates that, although surface concentrations of un-crosslinked PEI are lower compared to cross-linked PEI (Figure 7), a higher content of the NH2 groups exists on the uncross-linked PEI surface, resulting in higher reaction yields of HP. Multilayered Thin Films Obtained by Spin-Coating. Although one perhaps would anticipate concentration level differences between direct solution exposure (dipping) and spin-coating application methods of individual layers reacted to PVC, it would be rather surprising to detect differences in structural features of thin films. It appears that there are substantial changes in the reactions of HP, when spin-coating is elected as an application method. In the series of experiments discussed below, all thin films were spin-coated using the same concentrations and conditions as were utilized in direct solution exposure. When spin-coating was used to apply PEI and DS, no significant spectral differences were detected between both application methods, indicating that there are no structural differences among the thin films. The situation changed, however, when HP was spin-coated. Figure 10 illustrates ATR FT-IR spectra in the O-H bending region for HP applied by spin-coating. Trace A illustrates the spectrum of HP spin-coated obtained by using 0.005 g/mL

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Figure 11. ATR FT-IR spectra in the O-H stretching region for HP reacted with the PVC surface: (A) HP for the dipping condition; (B) HP at 23.8 s-1 for the spin-coating condition; (C) HP at 47.6 s-1 for the spin-coating condition; (D) HP at 60.2 s-1 for the spin-coating condition; (E) HP at 74.8 s-1 for the spincoating condition.

HP solution. It appears that a new band is detected at 1623 cm-1, which is attributed to the O-H bending modes of the HP-SO3-(H3O+) entities. As the HP solution concentration was increased from 0.005 (trace A) to 0.10 g/mL (trace E), the band intensity also increased. As we recall the results of the dipping experiments (Figure 5), when the HP solution concentration increased, the bands at 1650 and 1673 cm-1 were detected. These bands were attributed to the N-H deformation modes of the -CH2NH- linkages and unreacted PEI-NH3+(OH-) ionic complexes, respectively. However, for spin-coated HP, even though the HP concentration in the solution was increased, the bands due to formation of -CH2NH- linkages and unreacted PEI-NH3+(OH-) ionic complexes were not present. On the other hand, as shown in Figure 10, when HP is spin-coated, a new band at 1661 cm-1 attributed to the N-H deformation mode of PEI-NH3+SO3--HP ionic linkages is present. These observations indicate that there are significant differences in bonding characteristics of the HP molecules when dipping and spinning are employed. The primary difference between dipping and spincoating are shear rates and accessibility of HP molecules for reactions. Therefore, in an effort to identify how shear rates may affect HP reactions, the HP solution was spincoated using various shear rates. Figure 11 illustrates ATR FT-IR spectra in the O-H and N-H stretching region for HP on the PVC surface. For reference purposes, trace A represents the spectrum of HP immobilized using dipping. The band detected at 3571 cm-1 is attributed to the O-H stretching modes of HP-SO3-(H3O+).14 As seen, for spin-coated films, the 3571 cm-1 band disappears when high shear rates are applied (traces D and E). On the other hand, a broad band is detected around 2687 cm-1 and increases when shear rates are changed from 23.8 (trace B) to 74.8 s-1 (trace E). These observations indicate that HP immobilization occurs through the formation of ionic linkages between PEI-NH3+(OH-) and HP-SO3-(H3O+).4 When high shear rates are employed, the band

Kim and Urban

Figure 12. ATR FT-IR spectra in the O-H bending region for HP reacted with the PVC surface for various shear rates: (A) HP for the dipping condition; (B) HP at 5.9 s-1 for the spincoating condition; (C) HP at 23.8 s-1 for the spin-coating condition; (D) HP at 47.6 s-1 for the spin-coating condition; (E) HP at 60.2 s-1 for the spin-coating condition; (F) HP at 74.8 s-1 for the spin-coating condition.

Figure 13. Linear absorptivity of the N-H deformation band for HP reacted with the PVC surface for various shear rates: (A) HP for the dipping condition; (B) HP at 5.92 s-1 for the spin-coating condition; (C) HP at 23.81 s-1 for the spin-coating condition; (D) HP at 47.62 s-1 for the spin-coating condition; (E) HP at 74.83 s-1 for the spin-coating condition.

at 3462 cm-1 develops (traces D and E), which is attributed to the N-H stretching modes of newly formed -NH3+SO3-- ionic linkages. Although these observations suggest that HP is immobilized through the formation of ionic linkages between PEI-NH3+(OH-) and HP-SO3-(H3O+) groups when spincoating is applied, if this is indeed the case, the N-H deformation region should also confirm these findings. Figure 12 illustrates ATR FT-IR spectra in the N-H deformation region for HP immobilized to the pretreated PVC surface obtained by spin-coating under various shear rate conditions. For reference purposes, trace A is the spectrum of HP immobilized by dipping and exhibits the band detected at 1650 cm-1 attributed to the N-H deformation modes resulting from the -CH2NH- formation. On the other hand, the 1661 cm-1 band appears when a high shear rate is employed (traces E and F). As

Thrombresistant Multilayered Thin Films on PVC

Figure 14. ATR FT-IR spectra in the O-H bending region for HP reacted with the PVC surface: (A) TM polarization under the dipping condition; (B) TE polarization under the dipping condition; (C) TM polarization under the spin-coating condition; (D) TE polarization under the spin-coating condition.

indicated earlier, this band is attributed to the N-H deformation modes resulting from the formation of PEINH3+SO3--HP ionic linkages. When shear rates ranging from 5.9 to 74.8 s-1 are employed, while the 1661 cm-1 band increases, the 1650 cm-1 band significantly decreases. As shown in Figure 13, the linear absorptivity of the 1650 cm-1 band decreases from 1.30 × 10-3 to 0.06 × 10-3 cm-1 when the shear rate changes from 0 (dipping) to 74.8 s-1. These observations indicate that HP immobilization is indeed the shear rate dependent process, and at higher shear rates the formation of ionic linkages occurs. Orientation of HP Thin Films. Having identified that it is possible to control ionic or covalent bonding of HP molecules by changing shear rates of depositing, let us now consider orientation of HP molecules. It is well-

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known that the band intensities in IR are also affected by orientation of the electric vector responsible for a given vibration. Therefore, using polarized light, it is possible to estimate a preferential orientation of the surface species. Figure 14 illustrates ATR FT-IR spectra in the O-H bending region of the HP immobilized on the pretreated PVC surface obtained by spin-coating and solution exposure (dipping). While traces A and B illustrate transverse magnetic (TM) and transverse electric (TE) polarization spectra of specimens obtained by direct solution exposure (dipping), traces C and D illustrate the same polarization spectra of specimens produced by spinning. A comparison of the spectra of HP immobilized by direct solution exposure in TM (trace A) and TE (trace B) modes indicates that the enhanced intensity of the 1623 cm-1 band attributed to O-H bending modes is detected for TE polarization. In contrast, for spin-coated specimens, the TM polarization spectrum (trace C) exhibits a higher intensity, and the band at 1650 cm-1 is not detected. On the other hand, the 1661 cm-1 band appears. These observations indicate that the direct solution exposure generates the O-H groups of HP-SO3-(H3O+), which are preferentially parallel to the surface, thus providing evidence that the HP molecules are likely perpendicular to the surface. However, for the spin-coated specimens, the O-H groups of HP-SO3-(H3O+) are preferentially perpendicular, indicating that HP molecules are preferentially parallel. On the basis of these observations, the following structural features of HP orientation and bonding to the pretreated PVC surface are proposed: Upon direct solution exposure (0 shear rate), HP molecules are preferentially perpendicular, resulting from the formation of the -CH2NH- linkages between PEI-NH2 and HP-CHO groups. When spin-coating is employed, increased shear rates cause HP molecules to be oriented parallel to the surface, and for high shear rates, HP molecules are attached to the surface through the formation of ionic complexes between PEI-NH3+(OH-) and HP-SO3-(H3O+) groups. The proposed reaction mechanisms are depicted in Figure 15. Quantitative Analysis. The results of quantitative analysis of spin-coated films on the PVC surface are illustrated in Figure 16a where the surface concentration of PEI is plotted as a function of the PEI solution

Figure 15. Proposed reaction mechanism of HP immobilization through spin-coating.

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2.63 × 104 (3.02 × 102) to 4.78 × 10-4 mg/m2 (5.49 × 102 mg/m3). For spin-coated PEI, the surface concentration changes are shown in curve B. In this case, surface concentration levels are significantly lower, ranging from 5.58 × 10-5 (6.41 × 10) to 2.75 × 10-4 mg/m2 (3.16 × 102 mg/m3). The surface concentration changes of DS are shown in Figure 16b. Curve A illustrates the surface concentration of DS obtained by dipping, which ranges from 3.03 × 10-4 (3.44 × 102) to 7.18 × 10-3 mg/m2 (8.16 × 103 mg/m3), when the solution concentration varies from 0.005 to 0.1 g/mL. For spin-coated specimens, DS surface concentration ranges from 4.52 × 10-5 (5.14 × 10) to 4.70 × 10-4 mg/m2 (5.34 × 102 mg/m3) (curve B). Again, it is lower when compared to the thin films obtained by direct solution exposure. The effect of HP solution concentration on the HP surface concentration is shown in Figure 16c. Curve A illustrates surface concentration changes of HP immobilized on the PEI surface obtained by dipping and ranges from 6.90 × 10-3 (7.75 × 103) to 1.59 × 10-2 mg/m2 (1.79 × 104 mg/m3). For spin-coated specimens, the concentration levels are much lower, ranging from 1.26 × 10-4 (1.42 × 102) to 9.90 × 10-4 mg/m2 (1.11 × 103 mg/m3) for the same HP solution concentrations (curve B). Conclusions

Figure 16. (a) Surface concentration of PEI as a function of concentration for the spin-coating conditions: (A) PEI layer obtained by dipping; (B) PEI layer obtained by spin-coating. (b) Surface concentration of DS as a function of concentration for the spin-coating conditions: (A) DS layer obtained by dipping; (B) DS layer obtained by spin-coating. (c) Surface concentration of HP as a function of concentration for the spin-coating conditions: (A) HP obtained by dipping; (B) HP obtained by spin-coating.

concentration. For reference, curve A represents surface concentration changes of PEI obtained by dipping. As seen, when the PEI solution concentration changes from 0.005 to 0.1 g/mL, the surface concentration changes from

In this study, multilayered thin films that consist of PEI and DS were attached to PVC surfaces to immobilize HP. They were obtained using direct solution exposure and spin-coating methods. Although there are no significant differences between PEI and DS thin films, when direct solution exposure is employed as an application method, HP molecules become preferentially perpendicular to the surface, resulting in the formation of covalent linkages of -CH2NH- between PEI-NH2 and HP-CHO groups. On the other hand, for spin-coated thin films, HP molecules are preferentially parallel, thus immobilized through the formation of ionic linkages between PEI-NH3+(OH-) and HP-SO3-(H3O+) groups. Quantitative analysis indicates that the surface concentrations of each layer are significantly dependent on pH solution conditions. The concentration levels of HP immobilized to the modified PVC are significantly higher for specimens produced by direct solution exposure. Acknowledgment. The authors are thankful to the National Science Foundation Industry/University Cooperative Research Center in Coatings for support of these studies. LA980006Q