Platelet Compatibility Improvement by Proper Choice of Acidic

ARTICLE pubs.acs.org/Langmuir

Platelet Compatibility Improvement by Proper Choice of Acidic Terminal Functionality for Mixed-Charge Self-Assembled Monolayers Ching-Hsiung Shen and Jui-Che Lin* Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan 70101

bS Supporting Information ABSTRACT: In this study two different series of mixed-charge selfassembled monolayers (SAMs) prepared with
N+(CH3)3-terminated alkanethiol and strong dissociated monovalent
SO3H acid-terminated or weaker dissociated divalent
PO3H2 acidterminated alkanethiol in pure ethanol were characterized. The influence of the acidity of the anionic functionality in the mixedcharge SAMs on the surface characteristics and platelet compatibility was investigated. X-ray photoelectron spectroscopy indicated that a nearly equivalent amount of countercharged terminal groups was noted on the surface of
SO3H/
N+(CH3)3 mixed SAMs, while “
N+(CH3)3 thiol poor” phenomena were found on
PO3H2/
N+(CH3)3 mixed SAMs instead. This was caused by the distinct differences in solvation capability between the acidic anionic functional groups and solvent molecules and/or the interactions among the terminal ends of the thiols. This acidity difference also affected other interfacial properties and the platelet compatibility. The mixed SAMs formed from the mixture of
SO3H- and
N+(CH3)3-terminated thiols showed higher surface hydrophilicity and exhibited the least amount of platelets adhered, but these two mixed SAMs were all fairly negatively surface charged. The structure of the hydration layer near the surfaces was likely affected by the acidity of the anionic functionality, and this would cause such a distinct behavior in platelet compatibility. It was concluded that the hydrophilic surfaces with nearly equal amounts of surface positively and negatively charged components could exhibit better platelet compatibility. This work demonstrated that the nature of the acidic terminal ends of alkanethiol is also a key factor for preparing mixed-charge SAMs with good platelet compatibility.

1. INTRODUCTION Thrombus formation, caused by serial scenarios such as protein adsorption, platelet adhesion, and activation, may lead biomedical devices, such as artificial heart vessels, heart valves, and stents, to break down/fail.1,2 The interfacial interactions between the biological environment and the biomaterials that are controlled by the surface properties of the synthetic materials play important roles in thrombus formation.3,4 Therefore, pursuing the suitable surface properties of an artificial biomaterial with a better blood compatibility has become a research area of focus. Recently, self-assembled monoalyers (SAMs) prepared by longchain alkanethiol (HS(CH2)nX, n g 10) on gold can offer a densely packed, well-defined, and highly ordered surface with variant functional groups.5,6 Because of these outstanding advantages, this can serve as a model surface to study the blood
material interaction.7
12 For preparing mixed SAMs of alkanethiol, factors such as the nature of preparing the solvent and the type of terminal group of the alkanethiol, could influence the interfacial chemical properties of the SAMs formed.6,12
16 Moreover, the distinct interactions between the solvent molecules and terminal functionalities of the thiols and/or those among the tail groups of the thiols could also influence the kinetics of formation and the r 2011 American Chemical Society

mechanisms of the self-assembly process. Thus, the surface composition and spatial distribution of the resultant SAMs could be varied by changing the solvent and the type of alkanethiol used.12,14
17 Mixed-charge SAMs containing a mixture of cationic and anionic functionalities can exhibit excellent blood compatibility. This results from a surface with balanced charged groups.2,18
20 In addition to mixed-charge SAMs, protein nonfouling characteristics as well as good hemocompatibility properties were also reported on a surface grafted with a copolymer brush containing a mixture of cationic and anionic monomers or with a linear zwitterionic homopolymer.2,21,22 This further implicates that a surface with balanced charged groups is of potential use for biomedical applications. Therefore, how to control the parameter of the self-assembled process to prepare the mixed-charge SAMs with excellent nonfouling and biocompatible properties is important. Our previous study has shown that the nature of the solvent might affect the surface chemical properties for mixed SAMs composing the
NH2- and
SO3H-terminated thiol.12 However, the type of Received: September 4, 2011 Revised: November 20, 2011 Published: November 23, 2011 640

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cationic and anionic terminal groups, such as an anionic functionality with different dissociation constants (i.e., acidities), might also play roles to influence the properties of mixedcharge SAMs, but this is rarely discussed. In this study mixed-charge SAMs were prepared from a mixture of an alkanethiol with a positively charged monovalent terminal end (N,N,N-trimethyl(11-mercaptoundecyl)ammonium chloride) and an alkanethiol with different kinds of negatively charged acidic functionalities (11-mercaptodecanesulfonic acid and 11-mercaptoundecyl phosphonic acid) using pure ethanol as the solvent. The terminal group of 11-mercaptodecanesulfonic acid is a strongly dissociated “monovalent” acidic functionality, while the terminal group of 11-mercaptoundecyl phosphonic acid is a weakly dissociated “divalent” acidic end. The effects of these two acidic terminal ends on the interfacial properties and the platelet compatibility of the resultant mixed-charge SAMs prepared with a cationic
N+(CH3)3 terminal functionality in pure ethanol have been investigated. The results obtained indicate that the choice of acidic terminal ends could affect the surface and interfacial characteristics of the mixed-charge SAMs as well as their subsequent interactions with the biological environment.

Figure 1. Contact angle of
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 mixed SAMs as a function of the mole fraction of
N+(CH3)3 thiol in ethanol solution (XN+(CH3)3,soln). Data shown are the mean ( SD (n = 12). ethanol containing 1% HCl, and absolute ethanol. Then they were blown dry with nitrogen. 2.4. Surface Characterization. The surface wettability was characterized by the sessile drop technique (Face, model CA-A; Tokyo, Japan) at room temperature. The probing liquid was deionized water. The measurement was carried out at four different spots for each substrate, and the measurement at each spot was repeated three times. The surface composition measurement was carried out by X-ray photoelectron spectroscopy (XPS) or electron spectroscopy for chemical analysis (ESCA) (PHI Quantera SXM). The X-ray source was monochromatic Al Kα (hν = 1486.6 eV), and the takeoff angle was set at 45°. The high-resolution spectra were deconvoluted by mixing Gaussian
Lorentzian functions using the free software XPSPEAK. Quantification of the element was done on the peak areas of the C1s, O1s, N1s, P2p, and S2p multiplex. The ζ potential of flat surfaces was determined with a SurPass electrokinetic analyzer (Anton Paar KG, Graz, Austria). To mimic the physiological conditions, phosphate-buffered saline (PBS) at pH 7.4 was used as the electrolyte solution. When the electrolyte solution was flowed over the flat substrates under moderate pressure, the streaming potential was measured by two Ag/AgCl electrodes, placed at the inlet and outlet of the fluid cell. The ζ potential was calculated from the streaming potential, ΔUs, using the Helmholtz
Smoluchowski equation and Fairbrother
Mastin approach:25,26

2. MATERIAL AND METHODS 2.1. Synthesis of 10-Mercaptodecanesulfonic acid, 11mercaptoundecyl phosphonic acid, and N,N,N-trimethyl(11-mercaptoundecyl)ammonium Chloride. All reagents and solvents used were either HPLC or reagent grade and were used without further purification. The synthesis strategy of 10-mercaptodecanesulfonic acid was adopted from our previous published study.7 The 11mercaptoundecyl phosphonic acid and N,N,N-trimethyl(11-mercaptoundecyl)ammonium chloride were synthesized via a procedure from the literature.23,24 The structures of 10-mercaptodecanesulfonic acid, 11-mercaptoundecyl phosphonic acid, and N,N,N-trimethyl(11-mercaptoundecyl)ammonium chloride were characterized by NMR. 2.2. Preparation of Gold Substrates. The substrates used in this experiment were Si wafers and cover glass. The Si wafers were used in contact angle measurements, X-ray photoelectron spectroscopy (XPS), and in vitro platelet adhesion assay. The cover glass was used in ζ potential measurements. The Si wafers and cover glasses were separately cut into 3 cm  1.5 cm size and 5.5 cm  2.5 cm size, respectively. They were first cleaned with piranha solution (3:7 volume ratio of 30 wt % hydrogen peroxide solution and concentrated sulfuric acid) at 90 °C for 2 h. (Caution: The piranha solution is violently corrosive to organic matter and should be handled with great care!) Then they were thoroughly rinsed with a large amount of deionized water and absolute ethanol and blown dry with nitrogen. These cleaned substrates were immediately put into the vacuum chamber of a thermal evaporator for physical vapor deposition of Ti (Au adhesion promotion layer) and Au. The thicknesses of Ti and Au were 150 and 1000 Å, respectively.

ζ¼

ΔUs kη ΔP εε0

ð1Þ

where ΔUs/ΔP is the slope of the curve of the streaming potential vs increasing applied pressure through the streaming cell, η and ε are the viscosity and permittivity of the streaming electrolyte, ε0 is the permittivity of the free space, and k is the solution conductivity. 2.5. In Vitro Platelet Adhesion Assay. The procedures from our previous studies9
11 were followed and are briefly described below. The substrates were pre-equilibrated in Hepes
Tyrodes solution for 2 h. The human platelet-rich plasma was carefully added into the Petri dishes containing the specimen, and then the Petri dishes were placed inside the incubator with CO2 flow (5%) at 37 °C for 1 h. Following that, the substrates were gently rinsed with Hepes
Tyrodes solution three times and fixed with Hepes solution containing 2% (v/v) glutaraldehyde for 30 min. Then these substrates were rinsed sequentially with 100%. 75%,

2.3. Preparation of Mixed-Charge Self-Assembled Monolayers. The freshly prepared gold substrates were immediately immersed into two different mixed alkanethiol solutions containing a mixture of N,N,N-trimethyl(11-mercaptoundecyl)ammonium chloride with 10-mercaptodecanesulfonic acid or 11-mercaptoundecyl phosphonic acid. The solvent used for preparing the thiol solution was absolute ethanol, and the self-assembly condition was room temperature for 24 h. The mole fraction of
N+(CH3)3-terminated alkanethiol in mixed alkanethiol solutions was 0, 0.3, 0.5 0.7, and 1. The total concentration of the solution was set at 1 mM. After 24 h of immersion, these substrates were sequentially rinsed with absolute ethanol, absolute 641

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Table 1. Surface Composition (%)of
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 Mixed SAMs as a Function of the Mole Fraction of
N+(CH3)3 Thiol in Ethanol Solution (XN+Me3,soln)a
SO3H/
N+(CH3)3 mixed SAMs XN+(CH3)3,soln

a


PO3H2/
N+(CH3)3 mixed SAMs

C

O

S

N

C

O

S

N

P

0

69.11 (66.67)

22.85 (20.00)

8.04 (13.33)

0.00 (0.00)

67.54 (69.00)

23.75 (19.00)

3.05 (6.00)

0.00 (0.00)

5.66 (6.25)

0.3

74.40 (72.92)

14.22 (14.00)

7.04 (11.21)

4.34 (1.88)

70.05 (74.55)

19.66 (13.30)

3.38 (6.08)

2.13 (1.88)

4.78 (4.38)

0.5 0.7

74.54 (77.08) 74.00 (81.25)

14.57 (10.00) 14.75 (6.00)

7.29 (9.79) 7.77 (8.38)

3.60 (3.13) 3.48 (4.38)

69.15 (78.25) 71.05 (81.95)

19.72 (9.50) 18.52 (5.70)

3.48 (6.13) 3.17 (6.18)

2.55 (3.13) 2.93 (4.38)

5.10 (3.13) 4.60 (1.88)

1

81.53 (87.05)

10.53 (0.00)

4.22 (6.25)

3.72 (6.25)

81.53 (87.50)

10.53 (0.00)

4.22 (6.25)

3.72 (6.25)

0.00 (0.00)

Values listed in parentheses are calculated by the stoichiometric ratio of the alkanethiol within the solution.

Figure 2. S2p spectra of (a)
SO3H/
N+(CH3)3 and (b)
PO3H2/
N+(CH3)3 mixed SAMs as a function of the mole fraction of
N+(CH3)3 thiol in ethanol solution (XN+(CH3)3,soln).

noted on the S2p spectra of the
N+(CH3)3 SAM formed from 10% NH4OH ethanolic solution but not on the sample formed from pure ethanol (Figure S1 in the Supporting Information). This indicates that the
N+(CH3)3 SAM formed from pure ethanol can form a well-ordered monolayer. In contrast, the
N+(CH3)3 SAM formed from 10% NH4OH ethanolic solution contains disordered thiolate and/or a physically adsorbed alkanethiol mixed configuration that leads to the exposure of the terminal
SH group on the outer layer and, after oxidation, results in the formation of sulfonic acid as noted in the XPS measurement. Therefore, for preparing mixed-charge SAMs containing the
N + (CH 3 )3 -terminated thiol mixed with
SO3H-and
PO3H2-terminated thiols, pure ethanol was chosen as the solvent and 1% HCl ethanolic solution was used as the washing solution. 3.1. Contact Angle. Figure 1 shows the contact angle of
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 mixed SAMs

50%, and 25% Hepes
Tyrodes solution and deionized water and then dehydrated with 25%, 50%, 75%, and 100% ethanol for 3 min each with a shaker at 50 rpm. The substrates were dried with a CO2 critical point dryer (CPD) (Hitachi HCP-2, Japan) and immediately coated with a layer of Au. The densities of the adhered platelets were calculated from three different regions for each substrate at 1000 magnification. The platelet adhesion experiments were repeated three times for each sample.

3. RESULTS In this study 10% NH4OH ethanolic solution or pure ethanol was first chosen as the solvent for the preparation of the pure
N+(CH3)3 SAM. Then 1% HCl ethanolic solution was utilized for the washing step. Contact angle measurement showed the contact angle of the
N + (CH 3 )3 SAM formed from 10% NH4OH ethanolic solution (20.5 ( 2.38) was greater than that of SAM fromed from pure ethanol (8.0 ( 0.82). In addition, from XPS analysis, the sulfonic acid functionality (
SO3H) was 642

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as a function of the mole fraction of
N+(CH3)3-terminated thiol in ethanol solution (XN+(CH3)3,soln). On the
SO3H/
N+(CH3)3 mixed SAMs, the contact angle was lower than that of the pure
SO3H and
N+(CH3)3 SAMs, while the contact angle values were not significantly varied among the three mixed SAMs [θ(XN+(CH3)3,soln=0) > θ(XN+(CH3)3,soln=1) > θ(XN+(CH3)3, soln=0.3) ≈ θ(XN+(CH3)3,soln=0.5) ≈ θ(XN+(CH3)3,soln=0.7); oneway ANOVA followed by Tukey analysis; p < 0.05 if significant]. However, the
PO3H2/
N+(CH3)3 mixed SAMs had different trends. The contact angle of
PO3H2/
N+(CH3)3 mixed SAMs was higher than that of the pure
PO3H2- and
N+(CH3)3terminated SAM. Similar to the trend noted in
SO3H/
N+(CH3)3 mixed SAMs, the contact angle values among the three
PO3H2/
N+(CH3)3 mixed SAMs were not significantly varied [θ(XN+(CH3)3,soln=0.3) ≈ θ(XN+(CH3)3,soln=0.5) ≈ θ(XN+(CH3)3,soln=0.7) > θ(XN+(CH3)3,soln=0) > θ(XN+(CH3)3,soln=1); one-way ANOVA followed by Tukey analysis; p < 0.05 if significant]. In addition, the contact angle of the pure
SO3H SAM was greater than that of the pure
PO3H2 SAM. The mixed SAMs formed from the mixture of
SO3H- and
N+(CH3)3terminated thiols were more hydrophilic than those fromed from
PO3H2 and
N+(CH3)3 terminated thiols. 3.2. XPS. The surface atomic composition, as calculated by XPS of the
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 mixed SAMs, is shown in Table 1. For the
SO3H/
N+(CH3)3 mixed SAMs the surface atomic percentage of C1s, O1s, S2p, and N1s was almost the same except for the pure
SO3H and
N+(CH3)3 SAM. A similar finding was also noted for the
PO3H2/
N+(CH3)3 mixed SAMs, in which the surface atomic percentage of C1s, O1s, S2p, N1s, and P2p did not vary significantly except for the pure
PO3H2 and
N+(CH3)3 SAM. On the pure
N+(CH3)3 SAM (XN+ (CH3)3,soln = 1), oxygen was noted, and this might result from the adventitious oxygen-containing adsorbents. The results shown in Table 1 also indicate that the surface atomic percentages of S2p were all smaller than the theoretical values. This can be attributed to the inelastic scattering of the S2p photoelectrons from the thiolate binding that traveled through the monolayer from the near bottom of Au deposited into the detector.27 The S2p spectra of
PO3H2/
N+(CH3)3 mixed SAMs can be deconvoluted into two peaks, namely, the bound thiolate (162 eV) and unbound thiol (163.8 eV)28 (Figure 2b). After addition of the
SO3H-terminated thiol into the
SO3H/
N+(CH3)3 mixed SAMs, an additional peak appears at 168 eV, and it can be assigned to the sulfonic acid functionality (Figure 2a).7 Even as XN+(CH3)3,soln was increased, the area percentage of the sulfonic acid functionality remained similar for these
SO3H/
N+(CH3)3 mixed SAMs except for the pure
SO3H-terminated SAMs. The surface mole fraction of
N+(CH3)3 (XN+(CH3)3,surf) in these two mixed SAM series was calculated on the basis of the following equations:12,18 XNþ ðCH3 Þ3 , surf ¼

Figure 4. ζ potential of
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 mixed SAMs as a function of the mole fraction of
N+(CH3)3 thiol in ethanol solution (XN+(CH3)3, soln). Data shown are the mean ( SD (n = 3).

where AN+(CH3)3 is the normalized area of the trimethylamine functionality in the N1s spectra, ASO3H is the normalized area of the sulfonic acid functionality in the S2p spectra, and APO3H2 is the normalized area of the phosphonic acid functionality in the P2p spectra. From the correlation plot of the solution mole fraction of
N+(CH3)3-terminated thiol (XN+(CH3)3,soln) and the surface mole fraction of
N+(CH3)3 thiol (XN+(CH3)3,surf) (Figure 3), the
SO3H/
N+(CH3)3 mixed SAMs showed a sigmoid shape. In contrast, an “
N+(CH3)3 alkanethiol poor” phenomenon was noted on the
PO3H2/
N+(CH3)3 mixed SAMs. 3.3. ζ Potential. The ζ potential of
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 mixed SAMs as determined by the streaming potential in PBS, pH 7.4, was all negative except for the pure
N+(CH3)3 SAM (Figure 4). The ζ potential of the mixed SAMs formed from the mixture of
SO3H- and
N+(CH3)3-terminated thiol became gradually less negative with an increase of XN+(CH3)3,soln [ζ(XN+(CH3)3,soln=0) ≈ ζ

ðANþ ðCH3 Þ3 ÞXNþ ðCH Þ , soln 3 3

ðANþ ðCH3 Þ3 ÞXNþ ðCH Þ , soln þ ðASO3 H ÞXNþ ðCH Þ , soln 3 3

3 3

þ

ð2aÞ

for the
SO3 H=
N ðCH3 Þ3 mixed SAM XNþ ðCH3 Þ3 , surf ¼

Figure 3. Relationship between the surface mole fraction of
N+(CH3)3 (XN+(CH3)3,surf) and the solution mole fraction of
N+(CH3)3 thiol (XN+(CH3)3,soln) of
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 mixed SAMs.

ðANþ ðCH3 Þ3 ÞXNþ ðCH Þ , soln 3 3

ðANþ ðCH3 Þ3 ÞXNþ ðCH Þ , soln þ ðAPO3 H2 ÞXNþ ðCH Þ , soln 3 3

þ

for the
PO3 H2 =
N ðCH3 Þ3 mixed SAM

3 3

ð2bÞ 643

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pure
N+(CH3)3 SAM, was less activated (i.e., round and dendritic) than that of the adherent platelets on the Au control (Figure 6). Contrarily, the pure
N+(CH3)3 SAM showed a more activated morphology (i.e., dendritic spread), similar to that of the Au control.

4. DISCUSSION The nature of the solvent has been recognized as a primary factor controlling the surface chemical properties of mixed SAMs.6,15 A previous study showed that 3% triethylamine ethanolic solution was the best solvent to form a better packing quality of mixed SAMs prepared with alkanethiols terminated with cationic amine and anionic carboxylic acid functionalities.9 Moreover, the surface compositions of mixed SAMs prepared by
NH2- and
SO3H-terminated thiols could be modulated by the choice of solvent, such as DMSO and 10% NH4OH ethanolic solution.12 Holmlin et al. previously reported that the best solvent to self-assemble mixed SAMs with a 1:1 mixture of
N+(CH3)3- and
SO3H-terminated thiols was distilled deionized water.19 However, Chen et al. indicated that a basic aqueous (0.37% NH3 3 H2O) solution would be chosen to prepare mixed SAMs containing
N+(CH3)3 and
COOHterminated thiols.18 Therefore, the pH value of the solvent might influence the formation of SAMs with cationic functionalities such as
NH2 or
N+(CH3)3 terminal groups. On the basis of the contact angle measurement and XPS analyses, to form a good quality of mixed-charge SAMs containing an
N+(CH3)3-terminated thiol, pure ethanol was chosen as the solvent and a 1% HCl ethanolic solution was used as the washing solution. This further stresses the importance of the pH effect on the selfassembly process of cationic-functionality-terminated SAMs. In Figure 3 preferential adsorption for the
N+(CH3)3 thiol was observed on the
SO3H/
N+(CH3)3 mixed SAMs; even XN+(CH3)3,soln was less than 0.5. This is called “ideal nonideality” behavior in that the surface composition was independent of the solution composition and was similar to that of previous studies.12,18,29 However, on the
PO3H2/
N+(CH3)3 mixed SAMs the relationship between XN+(CH3)3,soln and XN+(CH3)3,surf became “surface
N+(CH3)3 thiol poor” instead. This was caused by the different solvation capabilities between the alkanethiol and solvent and/or the interactions among the terminal ends of the alkanethiols.12,14,15 Because the sulfonic acid functionality is a strongly dissociated monoacid while the phosphonic acid terminal end is a weaker dissociated divalent acid, variation in the hydrogen dissociation capability (i.e., acidity) between these two acidic terminal ends could likely lead to the distinct interactions among the terminal ends of the alkanethiol as well as those between the terminal functionalities and the ethanolic solvent molecules. Eventually the self-assembly process would be affected, and furthermore, the interfacial structure and the surface composition of these two series of mixed SAMs would be varied. The surface hydrophilicity of
SO 3 H/
N + (CH 3 )3 and
PO3H2/
N+(CH3)3 mixed SAMs showed different patterns with XN+(CH3)3,soln (Figure 1). The contact angle of the pure
SO3H SAM was greater than that of the pure
PO3H2 SAM. However, the mixed SAMs formed from the mixture of
SO3Hand
N+(CH3)3-terminated thiols were more hydrophilic than those formed from the mixture of
PO3H2- and
N+(CH3)3-terminated thiols. This might result from the different surface compositions and/or the nanoscale variation caused by the acidity of the anionic functionality that affected the self-assembly process.

Figure 5. Densities of adherent platelets on
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 mixed SAMs as a function of the mole fraction of
N+(CH3)3 thiol in ethanol solution (XN+(CH3)3, soln). Data shown are the mean ( SD (n = 3).

(XN+(CH3)3,soln=0.3) < ζ(XN+(CH3)3,soln=0.5) ≈ ζ(XN+(CH3)3,soln=0.7) < ζ(XN+(CH3)3,soln=1); one-way ANOVA followed by Tukey analysis; p < 0.05 if significant]. In contrast, the ζ potential did not vary significantly for the mixed SAMs formed from the mixture of
PO3H2- and
N+(CH3)3-terminated thiols except for the pure
N+(CH3)3 SAM [ζ(XN+(CH3)3,soln=0) ≈ ζ(XN+(CH3)3,soln=0.3) ≈ ζ(XN+(CH3)3,soln=0.5) ≈ ζ(XN+(CH3)3,soln=0.7) < ζ(XN+(CH3)3,soln=1); one-way ANOVA followed by Tukey analysis; p < 0.05 if significant]. The SAMs formed by the XN+(CH3)3,soln = 0 (i.e., pure
SO3H SAM) and XN+(CH3)3,soln = 0.3
SO3H/
N+(CH3)3 mixtures exhibited a more negative ζ potential than their counterparts formed by the
PO3H2/
N+(CH3)3 mixture. However, the XN+(CH3)3,soln = 0.5 and 0.7
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 mixed SAMs showed similar negative ζ potentials. 3.4. In Vitro Platelet Adhesion Assay. The densities of adherent platelets on
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 mixed SAMs are shown in Figure 5. On the
SO3H/
N+(CH3)3 mixed SAMs the least amount of platelets adhered was noted on the surface prepared from the XN+(CH3)3,soln = 0.3, 0.5, and 0.7 mixtures [N(XN+(CH3)3,soln=0) > N(XN+(CH3)3,soln=1) ≈ NAu > N(XN+(CH3)3,soln=0.3) ≈ N(XN+(CH3)3,soln=0.5) ≈ N(XN+(CH3)3,soln= 0.7); one-way ANOVA followed by Tukey analysis; p < 0.05 if significant). However, the densities of adherent platelets on the
PO3H2/
N+(CH3)3 mixed SAMs were increased with the addition of
N+(CH3)3-terminated alkanethiol but decreased on the pure
N+(CH3)3-terminated SAMs [N(XN+(CH3)3,soln=0.3) ≈ N (XN+(CH3)3,soln=0.5) ≈ N(XN+(CH3)3,soln=0.7) > N(XN+(CH3)3,soln=0) ≈ N(XN+(CH3)3,soln=1) ≈ NAu; one-way ANOVA followed by Tukey analysis; p < 0.05 if significant). Moreover, the platelet adhesion densities on the mixed SAMs prepared by the mixture of
SO3Hand
N+(CH3)3-terminated thiols was lower than those on the mixed SAMs prepared by the mixture of
PO3H2- and
N+(CH3)3-terminated thiols, but the XN+(CH3)3,soln = 0
SO3H/
N+(CH3)3 mixed SAMs (i.e., pure
SO3H SAM) adhered a greater amount of platelets than the XN+(CH3)3,soln = 0
PO3H2/
N+(CH3)3 mixed SAMs (i.e., pure
PO3H2 SAM). The morphology of the adherent platelets on
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 mixed SAMs, except the 644

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Figure 6. Representative SEM micrograph (2000) of adherent platelets on (a)
SO3H/
N+(CH3)3 and (b)
PO3H2/
N+(CH3)3 mixed SAMs as a function of the mole fraction of
N+(CH3)3 thiol in ethanol solution (XN+(CH3)3, soln).

The
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 mixed SAMs showed a negative ζ potential in PBS, pH 7.4, except the pure
N+(CH3)3-terminated SAM (Figure 4). The positive ζ potential noted on the pure
N+(CH3)3 SAM could be attributed to the terminal quaternized amine structure, which exhibited a cationic charge independent of the solution pH. The ζ potentials of the pure
SO3H and pure
PO3H2 SAMs were negative, and the pure
SO3H SAM had more negative surface charge than the pure
PO3H2 SAM. This was due to the difference in acidity of the anionic terminal ends, in which the sulfonic acid was a stronger dissociated acid than the phosphonic acid.

As for the mixed SAMs prepared by these two series of alkanethiol mixtures, the ζ potentials were all fairly negatively charged. Since the ζ potential is the surface charge at the slipping plane, it can be influenced by the surface chemical composition, surface polarity, and swelling behavior.30 Although these two mixed SAM series have different surface chemical compositions, the fairly negative ζ potential noted might be due to the similar ordering/orientation of ions or counterions in the slip plane. Detailed mechanisms remain to be determined. The adhesion densities and SEM morphology of the adherent platelets on
SO3H/
N+(CH3)3 and
PO3H2/
N+(CH3)3 645

dx.doi.org/10.1021/la203469b |Langmuir 2012, 28, 640–647

Langmuir mixed SAMs are shown in Figures 5 and 6. There were fair amounts of platelets adhered on the pure
SO3H and
PO3H2 SAMs, and the morphology of the adherent platelets was less activated forms. This finding was similar to those of our previous studies.7,8,11,12 However, the
N+(CH3)3 SAM that exhibited a positive charge also adhered a fair amount of platelets, but the morphology of the adherent platelets was more activated (dendritic spreading). This might result from the conformation variation of adsorbed proteins that further activated the adhered platelets. The platelet compatibilities on the mixed SAMs prepared with XN+(CH3)3,soln = 0.3, 0.5, and 0.7 for these two series of alkanethiol mixtures were quite distinct from each other. The mixed SAMs formed from the mixture of
SO3H- and
N+(CH3)3-terminated thiols exhibited the least amount of adherent platelets, while a fair amount of platelets remained adhered on the
PO3H2/
N+(CH3)3 mixed SAMs. Studies have presented that the adsorbed fibrinogen can mediate platelet aggregation and adhesion because of the specific receptor for the fibrinogen molecule, the glycoprotein IIB
IIIA (GP IIB
IIIA), on the platelet cell membrane.31,32 Chen et al. indicated that the mixed SAMs containing
N+(CH3)3- and
SO3H-terminated thiols (strong dissociated monovalent acid) are more resistant to fibrinogen adsorption than those containing
N+(CH3)3- and
PO4H2-terminated thiols (weaker dissociated divalent acid).18 Although the solvent used for preparing the SAMs in Chen et al.’s study18 and the current one are different (i.e., pH 7.4 PBS vs pure ethanol), their finding in protein adsorption might explain the observation that the mixed SAMs containing
N+(CH3)3- and
SO3H-terminated thiols had better platelet compatibility in this investigation. Likely owing to the different acidities of the anionic functionalities, the surfaces of
SO3H/
N+(CH3)3 mixed SAMs had similar surface amounts of cationic and anionic functionalities, independent of XN+(CH3)3,soln (i.e., XN+(CH3)3,surf ≈ 0.5) while the
PO3H2-terminated thiol was preferentially adsorbed on the
PO 3 H 2 /
N + (CH 3 )3 mixed SAMs. In addition, the
SO3H/
N+(CH3)3 mixed SAMs were more hydrophilic than the
PO3H2/
N+(CH3)3 mixed SAMs. Moreover, the more hydrophilic surface with similar surface amounts of anionic and cationic functionalities can exhibit better platelet compatibility. Previous studies have indicated that the surface with a crystalline layer with balanced charged groups or a zwitterionic linear polymer brush could have better blood compatibility owing to the charge balance effect and tightly bound water molecules on the topmost mixed SAMs.2,18,33 Therefore, the acidity of the anionic functionality could also influence the structure of the surface hydration layer for these two different series of mixed SAMs due to its effect on the surface charged group balance, subsequently resulting in different patterns in platelet compatibility. In summary, when preparing the mixed-charge SAMs, the acidity of the anionic functionality played the essential role in controlling the interfacial/surface characteristics and furthermore blood compatibility in vitro.

ARTICLE

prepared using pure ethanol as the solvent. XPS results indicated different surface composition patterns in the
N+(CH3)3terminated functionality were present. For the mixed SAMs formed from the mixture of
SO3H- and
N+(CH3)3-terminated thiols, XN+(CH3)3,surf was independent of XN+(CH3)3,soln and nearly about 0.5, but for the mixed SAMs prepared from the mixture of
PO3H- and
N+(CH3)3-terminated thiols, the “
N+(CH3)3 terminal end poor” phenomenon was shown. This might result from the different acidities of the anionic functionalities, which leads to distinct differences in solvation capability between the solvent molecules and terminal ends of the alkanethiol and/or the interactions among the terminal groups of the thiols. Furthermore, this acidity difference also influenced the surface wettability of these mixed SAMs. The mixed SAMs formed from the
SO3H- and
N+(CH3)3-terminated thiols exhibited higher surface hydrophilicity. However, these two types of mixed SAMs have similar fairly negatively charged ζ potentials in PBS at pH 7.4. The mixed SAMs containing the
SO3H- and
N+(CH3)3-terminated thiols exhibited better platelet compatibility in vitro than those containing
PO3H- and
N+(CH3)3terminated thiols. Therefore, the surfaces with nearly equal amounts of positively and negatively charged components and more surface hydrophilicity can reduce the platelet adhesion and activation. Hence, the nature of the acidic terminal group of the thiol is also a key factor affecting the interfacial properties and biomedical applications of the mixed-charge SAMs.

’ ASSOCIATED CONTENT

bS

Supporting Information. Figure showing the S2p spectra of the
N+(CH3)3 SAM formed from 10% NH4OH ethanolic solution and pure ethanol. This material is available free of charge via the Internet at http://pubs.acs.org/

’ AUTHOR INFORMATION Corresponding Author

*Phone: +886-6-275-7575, ext 62665. Fax: +886-6-234-4496. E-mail: [email protected].

’ ACKNOWLEDGMENT Financial support from the National Science Council, Taiwan, under Grants NSC 95-2221-E-006-305, NSC 97-2221-E-006017-MY3, and EZ-12-21-07-99 is gratefully appreciated. We thank the Tainan Blood Donation Center for providing human platelet-rich plasma. ’ REFERENCES (1) Rodrigues, S. N.; Goncalves, I. C.; Martins, M. C. L.; Barbosa, M. A.; Ratner, B. D. Biomaterials 2006, 27 (31), 5357–5367. (2) Zhang, Z.; Zhang, M.; Chen, S. F.; Horbetta, T. A.; Ratner, B. D.; Jiang, S. Y. Biomaterials 2008, 29 (32), 4285–4291. (3) Elbert, D. L.; Hubbell, J. A. Annu. Rev. Mater. Sci. 1996, 26, 365–394. (4) Roach, P.; Eglin, D.; Rohde, K.; Perry, C. C. J. Mater. Sci.: Mater. Med. 2007, 18 (7), 1263–1277. (5) Ulman, A. Chem. Rev. 1996, 96 (4), 1533–1554. (6) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105 (4), 1103–1169. (7) Lin, J. C.; Chuang, W. H. J. Biomed. Mater. Res. 2000, 51 (3), 413–423. (8) Tsai, M. Y.; Lin, J. C. J. Biomed. Mater. Res. 2001, 55 (4), 554–565.

5. CONCLUSION In this work two types of mixed SAMs containing quaternized
N+(CH3)3-terminated alkanethiols that were separately mixed with
SO3H (strong dissociated monovalent acid) and
PO3H2 (weaker dissociated divalent acid) terminated alkanethiol were 646

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