Article pubs.acs.org/Langmuir
Platelet Adhesion and Activation on Chiral Surfaces: The Influence of Protein Adsorption Yonghong Fan,†,‡ Rifang Luo,§ Honghong Han,†,‡ Yajun Weng,*,†,‡ Hong Wang,†,‡ Jing’an Li,†,‡ Ping Yang,†,‡ Yunbing Wang,§ and Nan Huang†,‡ † ‡ §
Key Laboratory of Advanced Technologies of Materials, Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610041, China S Supporting Information *
ABSTRACT: Adsorbed proteins and their conformational change on blood-contacting biomaterials will determine their final hemocompatibility. It has frequently been reported that surface chirality of biomaterials may highly influence their protein adsorption behavior. Here, lysine and tartaric acid with different chirality were immobilized onto TiO2 films respectively, and the influence of surface chirality on protein adsorption, platelet adhesion, and activation was also investigated. It showed that the L- and D-molecule grafted samples had almost the same grafting density, surface topography, chemical components, and hydrophilicity in this study. However, biological behaviors such as protein adsorption, platelet adhesion, and activation were quite different. The D-lysine grafted surface had a greater ability to inhibit both bovine serum albumin and fibrinogen adsorption, along with less degeneration of fibrinogen compared to the L-lysine anchored surface. However, the D-tartaric acid grafted surface adsorbed more protein but with less denatured fibrinogen compared to the L-tartaric acid grafted one. Further studies showed that the secondary structural change of the adsorbed albumin and fibrinogen on all surfaces with deduction of the α-helix content and increase of disordered structure, while the changing degree was apparently varied. As a result, the D-lysine immobilized surface absorbed less platelets and red blood cells and achieved slightly increased platelet activation. For tartaric acid anchored surfaces, a larger number of platelets adhered to the D-surface but were less activated compared to the L-surface. In conclusion, the surface chirality significantly influenced the adsorption and conformational change of blood plasma protein, which in turn influenced both platelet adhesion and activation.
1. INTRODUCTION Protein adsorption is a key event whenever a surface is exposed to blood plasma. The adsorbed plasma protein and its denaturation on the surface may initiate the coagulation cascade.1 A thin layer of albumin adsorption appears to minimize both the adhesion and aggregation of platelets, thus avoiding subsequent thrombus formation.2,3 It is well acknowledged that the conformation of adsorbed fibrinogen similar to that of the protein in solution does not activate the process of blood coagulation. In contrast, fibrinogen denaturation on biomaterials appears to be an indicator of fibrin network formation, promoting platelet activation, and resulting in severe blood clotting.4−7 Surface properties such as chemical components, wettability, and surface charge play a key role in the conformational change © XXXX American Chemical Society
that a protein undergoes upon adsorption on a biomaterial surface.8 Silva-Bermudez et al. reported different adsorption dynamics and orientations of the fibrinogen on different metal oxide surfaces.9 Steiner et al. studied the conformational change of fibrinogen adsorbed on hydrophilic and hydrophobic surfaces, respectively, using FT-IR spectroscopic imaging, and found that fibrinogen on hydrophilic surfaces retained its secondary structure, which might inhibit coagulation.10 Recently, research from Zhou et al. reported that despite the similar surface properties of both enantiomeric surfaces, the adsorption amount Received: July 2, 2017 Revised: September 2, 2017 Published: September 8, 2017 A
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at room temperature. At least five samples per group and three different areas per sample were measured and averaged in this experiment. Quartz crystal microbalances with dissipation (QCM-D) are often being used to monitor the process of molecular grafting and protein adsorption.15 After immersion into 2 mg/mL dopamine for 3 h twice and subsequent cleaning, the Au-coated single crystal sensor was inserted into the QCM chamber (Q-Sense AB, Sweden). Distilled water was injected into the channel to obtain a baseline first and the target molecule and Tris buffer were then pumped in successively at a speed of 50 μL/min. The variation of ΔF was recorded via Q-Soft, and the mass of grafted molecules was calculated via the Q-Tool. 2.4. Quantitative Determination of Protein Adsorption on the Surfaces. QCM-D was used to monitor the protein adsorption in this study. The chiral surfaces were prepared on Au-coated single crystal sensors as described above. Here, the PBS buffer was first pumped into the QCM-D’s channels to run a baseline. After the baseline got stable, the protein solution (BSA or Fib, 0.1 mg/mL) was injected at a speed of 50 μL/min. When the ΔF did not vary, the PBS buffer was subsequently introduced into the channels to remove weakly absorbed protein. The ΔF and mass variation were also obtained via Q-Soft and Q-Tool. The adhesion and denaturation of Fib were detected via enzymelinked immunosorbent assay. The platelet poor plasma (PPP) was gained via centrifugation (3000 rpm, 15 min) of fresh human whole blood (legally obtained from the Blood Center of Chengdu, China). Fib adsorption was evaluated by adding 50 μL of PPP onto the samples and incubation at 37 °C for 2 h.16 Then, samples were blocked with 1 mg/mL BSA after removing the PPP. The rabbit monoclonal anti-Fib (Sigma) was diluted 1:1000, and samples were incubated in the diluted solution for 1 h at 37 °C. Then the primary antibody was carefully rinsed with PBS thrice and a 1:3000 diluted secondary antibody (HRP conjugated mouse antirabbit IgG, Sigma) was added onto the samples and incubated at 37 °C for 1 h. After color development via the 3,3′,5,5′-tetramethylbenzidine (TMB) system, the solution was monitored by using a microplate reader at 450 nm. Fib activation was performed by merely replacing the primary antibody by γchain exposed sensitive primary antibody. 2.5. FTIR Analysis of Absorbed Proteins. Fourier transform infrared (FTIR, Nicolet 5700) spectroscopy was utilized to detect secondary structural changes of the adsorbed proteins. The spectra of original samples were detected first. Then these samples were immersed into a 1 mg/mL protein solution for 2 h and dried in a nitrogen atmosphere at 37 °C. After that, the protein adsorbed samples were scanned again. All spectra were taken by collecting 32 interferograms. The difference spectra were obtained using the protein adsorbed spectra subtract the original spectra of samples respectively, and the Fourier self-deconvolution was performed using the Omnic 2.1 software. Secondary structural analysis was done between the 1600−1700 cm−1 band of the original (no-smoothed) Fourier selfdeconvoluted amide I (amide I′) spectroscopy region according to previous publications.17 Gaussian curve-fitting was then performed via Peakfit v4.12 software after baseline correction (initial−final linear). The number and their peak positions, determined by second derivatization, were used as starting parameters. The curve fitting was stopped until the iteration arrived at 7 and r2 > 0.999. All component peaks were assigned to the specific secondary structure according to Langer et al.18 and Kroh et al.19 The secondary structure content was calculated from the proportion of fitted peaks to the total amide I′ band, and the adsorption-induced structural change was compared to the native state reported by Brandes et al.19 2.6. In Vitro Static Whole Blood Test and Platelet Adhesion. Whole blood was centrifuged at 1500 rpm for 15 min to separate the blood corpuscles, from which the platelet rich plasma (PRP) was obtained. Samples were put into a 24-well plate first. The static whole blood test was performed via addition of 400 μL of blood per well, while 50 μL of PRP was added onto the samples in the platelet adhesion experiment. After a 45 min incubation at 37 °C, all samples were twice washed with PBS and fixed in 2.5% glutaraldehyde for 12 h.
of fibrinogen, fibronectin, lysozyme, and human serum albumin on the L-cysteine assembled surface was larger than that on the D-cysteine assembled surface.11 In addition, Wei et al. investigated the adsorption of insulin on tartaric acid enantiomer anchored surfaces and found that insulin retained its bioactivity on the D-surface rather than the L-surface.12 The above two studies indicate that surface chirality may be a further important influencing factor of protein adsorption. However, few papers have been published about the influence of surface chirality on albumin adsorption or fibrinogen denaturation and the related platelet adhesion and activation. Hence, we suggest that the adsorption and denaturation of proteins (i.e., bovine serum albumin and fibrinogen) may be highly influenced by the surface chirality and are followed by differential platelet adhesion and activation on the enantiomeric surface. In this paper, lysine and tartaric acid with different chirality were grafted onto the biomaterial surface respectively; then, the influence of surface chirality on protein (bovine serum albumin and fibrinogen) adsorption was studied, and the platelet adhesion and activation were also evaluated.
2. MATERIALS AND METHODS 2.1. Materials. TiO2 films of the anatase phase were deposited using a UBMS450 high vacuum unbalanced magnetron sputtering system on the silicon (100) wafer; subsequently, they were cut into pieces with a size of 0.8 mm × 0.8 mm. L-Lysine, D-lysine, L-tartaric acid, D-tartaric acid, hexamethylene diamine (HD), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(N-morpholino)ethanesulfonic acid (MES), 3,4-dihydroxyphenylalanine (dopamine), tris-base (Tris), bovine serum albumin (BSA), and fibrinogen (Fib) from human plasma were all purchased from Sigma-Aldrich Chemical Co. Phosphate buffered saline (PBS, 0.01 M, pH = 7.4) and distilled water were also used for our experiments. 2.2. Preparation of Chiral Surfaces. The TiO2 films were successively washed with acetone, ethanol, and distilled water. Then the clean TiO2 substrates were placed into a cell culture dish and three layers of polydopamine films were deposited onto the TiO2 surfaces as previously described.13 For chiral lysine immobilization, the polydopamine coated samples were immersed into a 3 mM L- or D-lysine solution (Tris, pH = 8.5) for about 12 h at 37 °C, and lysine could be immobilized onto the polydoamine films either via Schiff base reaction or Michael addition.14 Because the tartaric acid could not be directly grafted onto the polydopamine films, HD, a small molecule with primary amine groups on both ends, was chosen as a linker to graft tartaric acid via a EDC/NHS/MES system (EDC/NHS/MES = 10 mM/6 mM/50 mM, pH = 5.5). After the polydopamine-coated samples were immersed into 5 mM HD solution (Tris, pH = 8.5) for about 12 h at 37 °C, the samples were put into the chiral tartaric acid solution (5 mM, activated via EDC/NHS/MES for 15 min) for 4 h. Finally, all lysine and tartaric acid grafted samples were cleaned with distilled water thrice to discard the unbounded molecules. The polydopamine coated, L- or D-lysine anchored, HD grafted, and L- or D-tartaric acid immobilized samples were named PDM, L-Lys, D-Lys, HD, L-TA, and D-TA, respectively. 2.3. Materials Characterization. X-ray photoelectron spectroscopy (XPS) analysis was performed using an X-ray photoelectron spectrometer (Thermo Fisher, USA) with a monochromatic Al Kα (hυ = 1486.6 eV) at 10−20 kV working voltage and 45 mA emission current. The barometric pressure within the chamber was set below 2 × 10−9 Torr, and the binding energy scale was corrected by setting the C1s peak to 284.6 eV. After careful drying in a vacuum drier and gold-coated, the samples were observed via field emission scanning electron microscopy (SEM, Quanta 200, FEI, Holland). Static water contact angles of the samples were conducted via sessile drop method on a DSA100 contact angle instrument (KRÜ SS, Germany) B
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Langmuir Next, the samples were rinsed with PBS thrice and rhodamine stained. Finally, they were photographed via inverted fluorescent microscopy (Zeiss, Germany) and examined via SEM.
(Table 1). Further study via QCM-D demonstrated that the same amount of chiral molecules was immobilized (Figure 1). Additionally, the carbon 1s (C1s) high-resolution spectra (Figure 2A) revealed a significant difference in the chemical components after chiral molecular grafting. The CO bond shifted into a lower binding energy (287.8 eV), caused by further immobilization of lysine and HD on PDM (288.4 eV). A new peak could be fitted to a component at 289.2 eV in the C1s spectrum after chiral lysine or tartaric acid grafting, which could be attributed to the −COOH group in both lysine and tartaric acid. As shown in Figure 2B, the result of the water contact angle revealed that there was no difference between L- and D-surfaces, indicating that the surface chirality did not influence the surface hydrophilicity under this condition. The surface topography was also obtained via SEM (Supporting Information, Figure S1), which also showed no significant difference between the L- and D-surfaces. 3.2. Effect of Surface Chirality on Protein Adsorption. In this study, BSA and Fib were chosen as model proteins to study the influence of surface chirality on plasma protein adsorption. QCM-D results are presented in Figure 3; the loss of frequency (ΔF) reflects the increase of the protein adsorption mass (mass change is shown in Supporting Information, Figure S2). This showed that the ΔF variation of L-Lys was higher than that of D-Lys both in BSA adsorption and Fib adsorption, indicating that more model proteins were absorbed on L-Lys. However, this phenomenon was reversed for the TA
3. RESULTS AND DISCUSSION 3.1. Immobilization of Chiral Molecules on Polydopamine Coating. The immobilization of chiral molecules on polydopamine coating was confirmed via XPS. Clearly, the atom percentage of carbon, nitrogen, and oxygen on L-Lys was nearly identical to that of D-Lys. The HD immobilization altered the surface of chemical compositions, as measured via a significant reduction in the content of oxygen and a slight increase in the contents of nitrogen and carbon, this result could be attributed to the lack of oxygen in HD. After tartaric acid was grafted on the HD, the content of oxygen increased again and nearly no difference of atom percentage was found for carbon, nitrogen, and oxygen between D-TA and L-TA Table 1. Elemental Compositions Obtained via XPS of PDM, HD, and Chiral Surfaces sample
C (%)
N (%)
O (%)
PDM HD L-Lys D-Lys L-TA D-TA
72.1 74.6 72.3 72.2 72.4 72.1
8.2 10.7 8.8 8.7 9.0 9.2
19.8 14.7 19.2 18.9 18.6 18.7
Figure 1. QCM-D analysis of chiral molecular grafting: (A) immobilization of L-lysine and D-lysine on polydopamine, (B) chiral tartaric acid grafting via the EDC/NHS/MES system.
Figure 2. Materials characterization of chiral surfaces: (A) C1s high resolution spectrum of samples, (B) water contact angle of samples. Please note that there are no significant differences between L- and D-surfaces. C
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Figure 3. QCM-D results showing protein adsorption on chiral surfaces: (A) BSA adsorption on L-Lys (left) and D-Lys (right), (B) Fib adsorption on L-Lys (left) and D-Lys (right), (C) BSA adsorption on L-TA (left) and D-TA (right), and (D) Fib adsorption on L-TA (left) and D-TA (right). The concentrations of BSA and Fib were 0.1 mg/mL, and the n insets show the overtone numbers of QCM-D.
of Fib was found on the L-surface rather than on the D-surface (Figure 4B). As there was no difference between L- and D-surface in the chemical component, surface topography, hydrophobicity, charge property, and functional groups, the differential protein adsorption might be attributed to surface chirality. The different surface chirality might either strengthen or weaken the
immobilized surface, where apparently the proteins preferred to be absorbed on D-TA rather than L-TA as shown in Figure 3B. The discrepant adsorption behavior of Fib on the chiral surfaces could also be observed in the ELISA result (Figure 4A). More amount of Fib adsorption caused a greater OD value at 450 nm, which agreed well with the QCM-D analysis. Moreover, an obviously increased denaturation D
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Figure 4. Fib adsorption and denaturation on chiral surfaces: (A) Fib adsorption and (B) Fib denaturation (the absorbance 450 nm reflects the amount of Fib adsorption or activation). Data are presented as mean ± SD (n ≥ 5), “*” shows p < 0.05, “**” shows p < 0.01, one-way ANOVA, compared between L- and D-surfaces.
interaction between biomaterials and proteins. The protein had a stronger interaction with chiral centers on the L-Lys than that on the D-Lys; thus, more proteins could be adsorbed on L-Lys, whereas the chiral centers on D-TA were more approachable for protein adsorption and result in more protein adsorption on D-TA compared to L-TA. Chiral interaction in a biological system is based on hydrogen bond, electrostatic interaction, and hydrophobic interaction, which can be strengthened or weakened by a matching degree of stereo structure. We suggest that the exposure of −NH2 groups on lysine immobilized surfaces and the −OH groups on the tartaric acid grafted surfaces in this experiment could influence the chiral interaction between chiral surface and proteins, leading to a different protein adsorption behavior on both pairs of chiral surfaces. 3.3. Secondary Structural Change of Absorbed Protein. To determine the secondary structure of the absorbed protein on chiral surfaces, FTIR spectra were collected respectively and the different spectra are shown in Supporting Information, Figure S3. The CO stretching of proteins is influenced by the environment, thus the overlapping component bands (α-helix, β-sheet, β-turn, and randomly coiled conformation) of the amide I band are closely related to the different types of secondary structure.20 Curve fitting of the Fourier self-deconvoluted amide I (amide I′) spectroscopy has been achieved (Figure 5). The position of fitting peaks was in good agreement with the second-derivative spectra, and 11 peaks were given in BSA or Fib curve fitting. All peaks were ascribed to CO vibration absorption except for the peak at 1611 cm−1 (this peak is present as the side chain of amino acid residues and benzene ring vibration absorption or selfassociation in Fib).20−22 These peaks were assigned to different secondary structures, and the secondary structure contents are summarized in Tables 2 and 3. As the α-helix and disordered structure were most frequently discussed, the change of the secondary structure was also considered in this experiment. The results showed that after being adhered onto the surfaces, the amount of α-helical structure in the BSA and Fib was strongly reduced; however, the amount of disordered structure increased more or less when compared with protein in 2H2O.19 This phenomenon was also reported by many researchers for other proteins.23−25 Specifically, the α-helix of BSA in 2H2O was reported to be about 53% but reduced to 43% on L-Lys, to 32% on D-Lys, 42% on L-TA, and to 49% on D-TA according to our findings (Table 2). Furthermore, a decrease of the α-helix in Fib after adsorption can be seen in Table 3 (31% in 2H2O, 30% on L-Lys, 24% on D-Lys, 17% on
Figure 5. FTIR analysis of secondary structures of BSA (left) and Fib (right) after adsorption (curve-fitted self-deconvoluted amino bond, from top to button: L-Lys, D-Lys, L-TA, and D-TA). The region of difference spectra (1600−1700 cm−1) was selected as the region of interest in the study of the secondary structure of proteins.
L-TA, and 27% on D-TA). Along with the decrease of the α-helix, an increase of disordered structure was observed. For BSA, the disordered secondary structure changed to 12% on L-Lys, to 15% on D-Lys, to 12% on L-TA, and to 11% on D-TA, as well as to 13% on L-Lys, to 12% on D-Lys, to 19% on L-TA, and to 11% on D-TA of Fib. The increments of the secondary structure are shown in Figure 6. Some researchers have already studied the secondary structural change of proteins on chiral surfaces. Ding et al.27 E
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Langmuir Table 2. Frequencies and Assignments of Amide I′ Components for BSA Adsorbed onto Chiral Surfaces sample L-Lys
L-TA
D-TA
in H2O (%)a
band frequency (cm−1)b
Gaussian curve fitting band position (cm−1)
area (%)
total (%)
area (%)
total (%)
area (%)
total (%)
area (%)
total (%)
β-sheet
33
1618−1638
1619 ± 1 1629 ± 3 1637 ± 2
7 7 5
19
8 12 17
37
7 10 10
27
4 8 4
16
disordered
8
1642−1648
1644 ± 1
12
12
15
15
12
12
11
11
α-helix
53
1651−1667
1652 ± 2 1659 ± 1 1667 ± 1
14 14 15
43
13 10 9
32
17 13 12
42
17 11 21
49
β-turns
5
1672−1695
1675 ± 2 1683 ± 2 1691 ± 3
10 11 5
26
6 7 4
16
11 6 2
19
13 6 5
24
assignments to secondary structure elements
a
D-Lys
2
These values are quoted from ref 19. bAssignments are based on refs 18,19,26.
Table 3. Frequencies and Assignments of Amide I′ Components for Fib Adsorbed onto Chiral Surfaces sample L-Lys
assignments to secondary structure elements
a
2
in H2O (%)a
band frequency (cm−1)b
β-sheet
37
1611−1638
disordered
10
α-helix
β-turns
Gaussian curve fitting band position (cm−1)
D-Lys
L-TA
D-TA
area (%)
total (%)
area (%)
total (%)
area (%)
total (%)
area (%)
total (%)
1620 ± 2 1627 ± 2 1635 ± 2
6 6 10
22
5 10 13
28
7 10 16
33
5 6 9
20
1640−1644
1643 ± 2
13
13
12
12
19
19
11
11
31
1651−1659
1651 ± 2 1659 ± 2
15 15
30
14 10
24
9 8
17
13 14
27 49
21
1663−1694
1666 1674 1682 1691
14 9 8 4
35
8 10 11 8
37
6 10 12 2
30
13 12 10 6
41
± ± ± ±
2 2 1 1
These values are quoted from ref 19. bAssignments are based on refs 18,19,26.
Figure 6. (A) Differences of the amide I′ components (%) between adsorbed BSA and BSA in solution. (B) Change in secondary structure element content (%) during adsorption of Fib from solution (curve fitted amide I′ band areas of adsorbed protein minus the same band in solution).
reported that the enzyme HRP immobilized on the L-NAsp PE surface showed better catalytic efficiency than that on D-NAsp PE, which the authors ascribed to the preservation of the secondary structure on L-NAsp PE surface (as analyzed via ATR-FTIR and CD). Wang et al.28 developed a gold nanoparticle
functionalized with D, L, or racemic penicillamine, respectively, to quantitatively analyze the adsorption and conformational features of transferrin. They suggested the existence of a significant difference between AuNP(D)-Tf and AuNP(L)-Tf systems in secondary structural change, which may have resulted F
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Figure 7. Immunofluoresent images of the whole blood test and platelet adhesion on chiral surfaces: (A) red blood cells and platelets adhesion in whole blood test, (B) platelet adhesion on chiral surfaces after incubation with PRP at 37 °C for 45 min. Quantitative measures of platelet or red blood cell adhesion: (C) the coverage ratio of platelet and red blood cells, (D) cell density of red blood cells, and (E) coverage ratio of platelet in platelet adhesion test. Data are presented as mean ± SD (n ≥ 5). “*” shows p < 0.05, “**” shows p < 0.01, “***” shows p < 0.001, one-way ANOVA, compared between L- and D-surfaces.
Figure 8. SEM images of red blood cells and platelet adhesion in (A) whole blood test and (B) platelet adhesion test.
protein adsorption of BSA and Fib might be the hydrogen bond and hydrophobic interaction.29 The surface chirality could influence the driving force, resulting in conformational matching between chiral centers and the proteins. It was easier for BSA or Fib to adhere onto L-Lys and D-TA according to QCM-D and ELISA results; however, lower increments were found in the α-helix change (see Figure 6) when compared to D-Lys and L-TA, respectively. This means that the interaction
from the different transferrin adsorption orientations. In their studies, a similar variation tendency of α-helix reduction and random coil increase of protein were observed after absorption on surfaces. While the residues on the surfaces and proteins were both negatively charged due to ionization in PBS, electrostatic interactions could not be beneficial to the protein adsorption in this study. Therefore, the main driving force for the differential G
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unactivated round morphology may not damage the hemocompatibility of biomaterials due to its reversibility and the adhered platelets can be desorbed from the surface. Activation of the resting platelets on the surface will initiate the irreversible clotting cascade. The FTIR analysis showed a significant increase in the disordered structure of Fib especially on L- Lys and L-TA. ELISA analysis confirmed that more Fib denatured on L- Lys and L-TA, which led to a higher platelet activation level on L- Lys and L-TA.
between L-Lys or D-TA and proteins was stronger than with its enantiomer immobilized surface. Another interesting phenomenon was also noticed while considering the results of Fib denaturation in combination with the change of disordered secondary structure. The Fib absorbed on L-Lys and L-TA followed a greater change in the disordered structure, which in turn led to a conformational change of the protein peptide chain, thus exposing the γ-chain in Fib. As a result of γ-chain eversion, Fib can be easier activated by thrombin, thus promoting platelet adhesion and activation behaviors. In summary, the FTIR analysis indicated that the surface chirality significantly influenced the secondary structural change of blood proteins and further influenced the hemocompatibility. 3.4. In Vitro Static Whole Blood and Platelet Adhesion Test. Because red blood cell adhesion and platelet activation behaviors are very important to blood contact materials, static whole blood and platelet adhesion tests were also performed. The results below (Figure 7) show that much fewer red blood cells were found to adhere onto the D-surfaces rather than on L-surfaces. The coverage ratio of platelets on D-Lys was far lower than that on L-Lys. In contrast, the platelet coverage on D-TA was higher than that on L-TA. Further investigation via SEM (Figure 8) showed the details of platelet activation. The adhered platelets could be classified to five different morphologies, i.e., round, dendritic, spread dendritic, spreading, and fully spreading shapes,30 and the activation level increased gradually according to the change in shape. The platelets on D-Lys presented as round and dendritic; however, some spread dendritic on L-Lys. Noticeably, the adhered platelets on D-TA were much more than those on L-TA, but most of the platelets had fully spreading shapes on L-TA, which indicates a lower activation level of D-TA (Figure 9).
4. CONCLUSIONS In summary, the chiral molecule anchored surfaces had almost the same grafting amount, surface topography, chemical component, and hydrophilicity while inducing different protein adsorption behaviors. More BSA and Fib were adsorbed on surfaces of L-Lys and D-TA, which contributed to more platelet adhesion. Less Fib denaturation (exposing the γ-chain) and the secondary structural change (increase of random coil) on surfaces of D-Lys and D-TA reduced the platelet activation degree compared to that of the L-ones. Therefore, these results indicate that surface chirality can strongly influence both protein adsorption and platelet adhesion, which provides clues for blood contact material design.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b02283. Surface topography of samples measured by SEM; protein adsorption on chiral surfaces monitored by QCM-D, the mass increase of absorbed protein BSA and Fibon chiral lysine immobilized surfaces, BSA and Fib on chiral TA grafted surfaces; difference spectra of protein absorbedchiral surfaces BSA, Fib (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Address: Key Laboratory of Advanced Technologies of Materials, Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China. ORCID
Yonghong Fan: 0000-0002-5132-0499 Jing’an Li: 0000-0002-0305-6929 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The financial support for this work by National Natural Science Foundation of China (No. 31270020), Sichuan Province Science Foundation for Youths (No. 2013JQ0043), China Postdoctoral Science Foundation(2017M612967) and Fundamental Research Funds for the Central Universities (2682016YXZT14, 2682014CX006) is greatly acknowledged.
Figure 9. Statistic analyses of platelet morphologies on samples. Data are presented as mean ± SD (n ≥ 5). “*” shows p < 0.05, “**” shows p < 0.01, “***” shows p < 0.001, one-way ANOVA, compared between L- and D-surfaces.
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QCM-D and ELISA results suggest that L-Lys and D-TA had a greater ability to absorb both BSA and Fib compared to that of D-Lys and L-TA, respectively, while less Fib denaturation appeared on D-Lys and D-TA. It was reported that more BSA adsorbed or coated on the surface may reduce platelet adhesion;2 however, in these cases, adsorption of Fib also decreased. When both adsorption of BSA and Fib increased, it seemed that the amount of adsorbed Fib rather than albumin determined the activation of platelets. Platelet adhesion with
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DOI: 10.1021/acs.langmuir.7b02283 Langmuir XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.langmuir.7b02283 Langmuir XXXX, XXX, XXX−XXX
