Bioinspired Multiple-Interaction Model Revealed in Adsorption of Low

Jun 6, 2013 - BioSciences Deptartment, Fresenius Medical Care Deutschland GmbH, Bad ... precipitation (HELP),5 double filtration,6 and direct adsorpti...
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Bioinspired Multiple-Interaction Model Revealed in Adsorption of Low-Density Lipoprotein to Surface Containing Saccharide and Alkanesulfonate Jing Li,† Xiao-Jun Huang,*,† Jörg Vienken,‡ Zhi-Kang Xu,† and Thomas Groth§ †

MOE Key Laboratory of Macromolecular Synthesis and Functionalization (Ministry of Education), Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China ‡ BioSciences Deptartment, Fresenius Medical Care Deutschland GmbH, Bad Homburg 61352, Germany § Biomedical Materials Group, Deptartment of Pharmaceutics and Biopharmaceutics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale) 06120, Germany S Supporting Information *

ABSTRACT: A new “multiple-interaction model” for low-density lipoprotein (LDL) adsorption to a specific surface containing saccharide and alkanesulfonate ligands is proposed. The model suggests that there are interactions of the saccharide component beyond electrostatic interactions of the alkanesulfonate component that both influence the LDL adsorption process. This concept of multiple interactions between saccharide and LDL was inspired by the similarity in structures of LDL receptors (LDLR), heparin, and heparans used in LDL-apheresis. The model was confirmed by SPR analysis by the adsorption maxima on SAM surfaces with different compositions of saccharide and alkanesulfonate and additionally by CD detection of the conformation of LDL when in contact with saccharide.

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INTRODUCTION An abnormally high level of plasma low-density lipoprotein (LDL) in human plasma is a key pathogenic factor that contributes to atherosclerosis and finally leads to coronary artery diseases (CAD).1 For over 25 years, a variety of LDLreduction therapies have been established. Extracorporeal LDL apheresis systems have proven to be particularly effective for the therapy of familial hypercholesterolemia (FH).2 These systems include immunoadsorption (IMA),3 dextran sulfatecellulose adsorption (DSA),4 heparin extracorporeal LDL precipitation (HELP),5 double filtration,6 and direct adsorption of lipoprotein (DALI).7 Moreover, adsorbents in different apheresis systems play important roles in the elimination of LDL in blood, such as sulfonated polysaccharides in DSA,4 heparin molecules in HELP,5 and poly(acrylic acid) in DALI7 systems. Under physiological conditions, ligands applied in the adsorbents are all rich in acidic groups such as −SO3− and −COO−, because according to current belief that electrostatic interactions between positively charged regions of apolipoprotein-B100 (apoB100) in LDL and negatively charged ligands of the adsorbent affect the adsorption of LDL onto the adsorbents8 and therefore result in highly efficient removal of LDL during plasma or blood cleansing processes. However, for years another important aspect has been neglected: most of the adsorbents mentioned above are saccharides or saccharide-like structures. They offer similar structures to that of the natural LDL-binding glycoprotein, © 2013 American Chemical Society

LDL receptor (LDLR). As is well-known, LDLR consists of three main domains: LDL receptor type A (LA) repeats, epidermal growth factor precursor (EGFP), and O-linked oligosaccharides.9 Interestingly, the exact physiological function of this O-linked oligosaccharide domain is still poorly understood10 due to the extraordinary complexity of LDL and LDLR. The similarity of the saccharide components between LDL-adsorbent and LDLR stimulated us to outline the hypothesis that those saccharides on the ligands can be efficiently used for improving the adsorption behavior of LDL with adsorbents. In our previous work we aimed at improving the adsorption efficiency of biomaterial surfaces11 and simplify their fabrication process,12 i.e., of heparin-modified polysulfone (PSf) membranes under various technological conditions for application in hemodialysis with simultaneous LDL removal.13 This work deals with a new perspective on the chemical composition of adsorbents to be used for extracorporeal LDL-apheresis based on the observation that the saccharide components take part in the LDL adsorption process. A mechanism has been proposed based on the “multiple-interaction model” (Figure 1), of which experimental details are provided in this paper. This new concept may provide new ways to study the interaction Received: April 17, 2013 Revised: June 3, 2013 Published: June 6, 2013 8363

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Table 1. Preparation of Mixed-Thiol SAM Surfaces of Different Compositions on SPR Chipsa solution ratio

type of SAM produced

1:0 5:2 1:1 2:5 0:1

glycolized SAM (1−0) mixed-thiols SAM (5−2) mixed-thiols SAM (1−1) Mixed-thiols SAM (2−5) MPS SAM (0−1)

a Mixed-thiol solutions were blended with different solution ratios of 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside thiol (glycolized)/DMF (1 mM) and sodium 3-mercapto-1-propanesulfonate (MPS)/ethanol (1 mM). SPR chips were then deacetylated in a solution of sodium methylate/methanol diluted in methanol at 0.1 M, in shaking bath, at around 30°C for at least 6 h. After the deacetylation reaction (which de-protected the hydroxyl groups of the assembled saccharide), rinsing cycles and drying were performed.

solution ratios of 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside thiol/ N,N-dimethyl formamide (DMF) (1 mM) and sodium 3-mercapto1-propanesulfonate (MPS)/ethanol (1 mM). SPR chips were immersed into the solution for at least 18 h at room temperature to allow SAM formation.17 After that, the SPR chips with different surface compositions were rinsed three times with a DMF/ethanol solution (v/v = 1:1) followed by water to remove any unbound thiol molecules. After rinsing cycles, the SPR chips were dried in nitrogen. Characterization of Different Modified SPR Chips. X-ray Photoelectron Spectroscopy (XPS) Detection. X-ray photoelectron spectroscopy (XPS) is a quantitative spectroscopic technique that measures the elemental composition, empirical formula, chemical state and electronic state of the elements that exist within a material. XPS experiments were carried out on a RBD Instruments upgraded PHI5000C ESCA system (Perkin-Elmer) with Al Kα radiation (hν = 1486.6 eV). The base pressure of the analyzer chamber was about 5 × 10−8 Pa. Both full-range spectra and narrow spectra with a high resolution for all the elements were recorded by using an RBD 147 interface (RBD Enterprises, USA) with AugerScan 3.21 software. Binding energies are calibrated with respect to the bulk Au 4f7/2 peak at 84.00 eV. Atomic Force Microscope (AFM) Imaging. Atomic force microscopy (AFM) (SPI3800, Seiko Instruments Inc., Japan) was used to study the surface morphology of different modified SPR chip surfaces. The AFM observation was performed under nitrogen protection at room temperature with a 20-μm scanner in tapping mode. All images were collected in the “height mode” of the SPI3800 and were taken at a scanning speed of 1 Hz. Static Water Contact Angle (WCA) Measurement. The hydrophilicities of SPR chip surfaces with different compositions are characterized by water contact angle assessments. Static contact angle was measured at room temperature on a CTS-200 contact angle goniometer (Cellcons Controls, China) equipped with video capture. Static contact angles were measured by sessile drop method. A 2 μL drop of water was set onto chip surfaces with a microsyringe. Digital images for the droplet were then recorded. Contact angles were calculated from these images with software. Each reported value was an average of at least five independent measurements. Surface Plasmon Resonance (SPR) Analysis of Dynamic Protein Interactions with Modified Surfaces. Surface plasmon resonance (SPR) is a valuable tool to investigate biological interactions.18 SPR offers real time in situ analysis of dynamic surface events and is capable of defining rates of adsorption for a range of surface interactions. All SPR measurements were performed with a SR7000DC instrument (Reichert, USA). Different concentrations (1, 5, 10, 15, 20, 25, and 30 μg/mL) of LDL in PBS (phosphate buffered saline: 137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, pH 7.4) solutions were injected accordingly into tubes and flowed over SPR chip surfaces at a rate of 25 μL/min at 25.0 ± 0.1 °C. Injection was maintained for 300 s to allow protein adsorption, and then buffer solution was automatically injected to complete one

Figure 1. Multiple-interaction model for LDL in contact with adsorbents containing negative ligands and neutral saccharides. It is assumed that there are multiple saccharide−LDL interactions as well as electrostatic interactions.

between LDL and LDLR,14 e.g., defining the relationship between diabetes and hyperlipidemia,15 or to design more efficient and low cost LDL adsorbers.16 To confirm the “multiple-interaction model”, the interactions between LDL protein and several model surfaces constructed with different proportions of saccharide and alkanesulfonate ligands are examined. Surface plasmon resonance (SPR) is applied to investigate the dynamic adsorption processes between protein and adsorbents, and the circular dichroism (CD) technique is used to detect secondary structure changes of proteins associated with free saccharides in solution.

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EXPERIMENTAL SECTION

Materials. 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranoside thiol was obtained from Chemsynlab Pharmaceutical Science & Technology, Beijing, China. Sodium 3-mercapto-1-propanesulfonate (MPS) was a commercial product of Aladdin Reagent, Shanghai, China. Low density lipoprotein (LDL) was purchased from Millipore, Billerica, USA. Sodium methylate/methanol and lecithin were purchased from Sinopharm Chemical Reagents, Shanghai, China. All other reagents were supplied by Sinopharm Chemical Reagents, Shanghai, China. SPR chips (Au chip) were provided with SPR equipment as a complete set from Reichert, USA. The water used in all experiments was deionized and ultrafiltered in order to obtain a resistance of 18 MΩ·cm with a TKA MicroPure Water system. Preparation of Model Surfaces with Different Compositions. Prior to the formation of self-assembled monolayers (SAM), bare SPR chips (Au chip) were carefully cleaned in order to remove possible contaminants. After prewashing with acetone followed by rinsing with deionized water in an ultrasonic cleaner, the bare SPR chips were treated with piranha solution (H2SO4/H2O2 = 7:3) for around 5 min, followed by a rinsing process with copious amounts of deionized water. The precleaned bare SPR chips were finally dried under nitrogen before being immersed immediately into different SAM reagent solutions. SPR chip surfaces of different compositions, containing saccharides and negative ligands, could be prepared via a two step process: creation of mixed-thiol SAMs (Table 1) followed by deacetylation reaction. A series of mixed-thiol solutions was blended with different 8364

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Figure 2. XPS narrow, high resolution, spectra of elements O 1s and S 2p3. Curves for five SPR chips prepared with different input ratios of SAM solutions are shown. SPR chip surfaces with different input ratios of SAM solutions would be expected to have correspondingly different surface ratios of O/S. injection procedure for one protein concentration. PBS (pH 7.4) was used as the buffer solution for the whole analysis process. Lecithin in ethanol (0.1 g/mL) was introduced as a blocking agent to prevent hydrophobic interactions of proteins to chip surfaces. Circular Dichroism (CD) Spectroscopy for Protein Secondary Structure Analysis. Circular dichroism (CD) spectroscopy is a currently used method in structural biology for the examination of proteins, polypeptides, and peptide structures since the 1960s.19 Circular dichroism spectra from the different LDL samples are recorded on a spectropolarimeter (MOS-450 AF/AF-CD, Bio-Logic, France) using a 0.2 cm quartz cuvette. PBS (pH 7.4) is used as buffer to simulate physiological conditions for the protein. For three LDL samples, LDL (100 μg/mL) is diluted in PBS (pH 7.4), Glc/PBS (0.1 M glucose in PBS (pH 7.4)), and Gal/PBS (0.1 M galactose in PBS (pH 7.4)). All CD spectra of LDL were obtained only after subtracting their corresponding background absorptions of different buffer systems. The cell holder compartment is maintained under a nitrogen atmosphere at room temperature. Data from at least five spectra are taken as an average.

Table 2. Accurate Surface Ratios of Glucosyl to Sulfonic Groups Calculated from the Integration of XPS Peaks of O 1s and S 2p3 Respectively (from the Spectra in Figure 3) sample

area of O 1s

area of S 2p3

ratio of O/S

compositiona

(1−0) (5−2) (1−1) (2−5) (0−1)

× × × × ×

× × × × ×

4.13 3.65 3.44 3.28 3.07

1−0 1.20 0.54 0.25 0−1

2.92 3.12 3.28 3.38 3.56

4

10 104 104 104 104

0.71 0.86 0.95 1.03 1.16

4

10 104 104 104 104

a

The actual molar ratio of glucosyl to sulfonic groups for each modified SPR chip surface.

of typical AMF images of surfaces produced using different SAM solution ratios are shown in Figure 3. The coefficient Ra

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RESULTS AND DISCUSSION This research centered on the effects of saccharide-moieties in ligands during the LDL adsorption process, in an attempt to confirm the existence of a specific saccharide−LDL interaction, i.e., to verify the proposed multiple-interaction model. To eliminate unnecessary influencing factors, model surfaces containing both glucosyl and propylsulfonic groups were established on Au SPR chips by mixed-thiol SAM modifications. Characterization of Different Compositions of Model Surfaces. The exact surface composition was accurately detected by X-ray photoelectron spectroscopy (XPS; Figure 2). The narrow, high resolution spectra of elements O 1s and S 2p3 for five SPR chips of different compositions were analyzed and are depicted in Figure 2. Obviously, the assembled β-Dglucopyranoside thiol and sodium 3-mercapto-1-propanesulfonate contain different ratios of O/S; therefore, SPR chip surfaces with different input ratios of SAM solutions would be expected to have correspondingly different surface ratios of O/ S. Accurate surface ratios of O/S could be calculated from the position and integration of XPS peaks, and thus the exact surface composition ratios of glucosyl groups to sulfonic groups could be obtained (Table 2). These ratios differed from the ratios of input solutions, probably because of different binding efficiencies to the Au surface of thiols with different molecular sizes; smaller, linear thiols tend to form SAMs more easily.20 Subsequently, the morphologies of the modified SPR chips were characterized by atomic force microscopy (AFM). A series

Figure 3. Atomic force microscopy images (2 μm × 2 μm) and roughness coefficients (Ra) of different modified SPR chip surfaces. Input ratios of SAM solutions are given in white parentheses. The coefficients Ra (nm), indicating the degree of surface roughness, differ obviously according to the different proportions of alkanesulfonate.

(nm) indicates the degree of surface roughness of different modified SPR chips. The values of Ra all increased significantly as the mixed thiols were added and the mixed SAMs were formed. When the proportion of alkanesulfonate increased, the values of Ra suggested a slight increase, which meant the higher roughness with the smaller molecular size of the surface 8365

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immobilized alkanesulfonate compared to saccharide. This might be attributed to the electrostatistic repulsion between the alkanesulfonate molecules during their self-assembling process, and it resulted a farther distance of the surface assembled molecules thus performed as a higher surface roughness. The static water contact angle (WCA) was used to estimate the hydrophilicities of the SPR chip surfaces of different compositions as preliminary characterization of the mixed-thiol SAM formation step.17 Those detection surfaces involved were prior to the deacetylation procedure, which meant that the mixed-SAM surfaces for the WCA test were consisted of acetylated saccharide and alkanesulfonate. Since the hydroxyl groups of the applied saccharide (2,3,4,6tetra-O-acetyl-β-D-glucopyranoside thiol) were preprotected with acetic anhydrides, the assembled saccharide was hydrophobic before deacetylation, while the other component, containing sulfonic groups, exhibited hydrophilicity. Therefore, the degree of hydrophilicity correlated with the ratio of sulfonic ligand to acetylated saccharide (Figure 4). The higher contact

Figure 5. Typical SPR adsorption sensorgram for sample (5−2), i.e., mixed thiol SAM surface with composition ratio of approximately 1.20 glucosyl to sulfonic groups. The injection time was fixed at 300 s before buffer was injected. When protein solutions injected into, an increase in the SPR detected response (μRIU) was induced due to protein adsorptions onto chip surfaces; while when buffer injected, SPR response declined because of reversible desorption of protein.

system automatically changed to buffer, resulting in a slight decline in the response signals. That decline could be attributed to the washing out of reversibly adsorbed LDL from the SPR chip surfaces by the buffer. With increasing concentration of LDL solution injected, the adsorption curve gradually exhibits a tendency toward saturation. The adsorption kinetics of the protein−ligand interaction processes could be analyzed from the key factor, i.e., the rate of adsorption. This can be obtained from the steepest positive gradient of the SPR adsorption profile. This rate is dependent both on the intrinsic kinetics and the mass transport of the system. Normally the process of diffusion of the adsorbate to the surface is generally much slower than the intrinsic adsorption kinetics and is thus the rate-determining factor.18 However, the mass transport was negligible in this system, because of the extremely thin SAM layer on the SPR chips. Therefore, the Langmuir isothermal adsorption model21 was applied for simulation of the LDL adsorption kinetics. The SPR response change at equilibrium was related to the protein concentration according to the following equation: 1 1 1 1 = + R eq R maxKA C R max (1)

Figure 4. Hydrophilicity of mixed-thiol SAM surfaces with different compositions. The higher contact angle of sample (1−0) suggests a SAM of acetylated saccharide, while the lower CA of other samples (5−2, 1−1, 2−5) suggests that the sulfonic ligand of the mixed thiol has been incorporated. (Au-surface used as a control).

angle (CA), i.e., 57.5° for sample (1−0) compared to 48.3° for the bare Au surface (control), suggested the formation of a SAM of acetylated saccharide, while the lower CA values of other samples (5−2, 1−1, 2−5), ranging from 20−25°, suggested incorporation of the sulfonic ligand of the mixed thiol. The lowest CA of sample (0−1), around 17.6°, corresponded to the homo MPS SAM. These differences in hydrophilicity suggested that the mixed-thiol SAM process was successfully applied in the formation of model surfaces with mixed functional groups. Dynamic Analysis of Protein Interactions by Surface Plasmon Resonance (SPR). Surface plasmon resonance is rapidly gaining recognition and application as a powerful tool for biomaterial characterization, as it is able to rapidly monitor any dynamic process, such as adsorption, in real time without the need to label the adsorbate.18 A typical SPR adsorption curve for sample 5−2 is shown in Figure 5 (not shown is the blocking pretreatment). When LDL protein solutions with different concentrations were injected into SPR tubes, proteins in the flow would interact with the SPR chips modified with saccharide and alkanesulfonate (fixed in the channel), which would induce an increase in the SPR detected response (μRIU). After the injection process for 300 s, the instrument

where Req is the SPR response to the injection of LDL with concentration of C at equilibrium and Rmax is the equilibrium response when C is infinity. The linear fit of the Langmuir adsorption process is illustrated in Figure 6, with the relationship obtained being y = 2.19 × 10−4 + 4.54 × 10−13x (R = 0.9993). The value of KA obtained by SPR technique for LDL binding with the sample (5−2) surface was calculated from the y intercept and gradient to be 4.82 × 108 M−1 (Figures 5 and 6). The purpose of this paper was to study the influence of saccharides on adsorption process of LDL diluted in PBS. Therefore, the comparison of LDL adsorption amounts (for a constant protein concentration) among chips with different surface saccharide to alkanesulfonate group ratios is especially important. The histogram of the adsorptions of LDL for the different adsorbents is plotted in Figure 7. It is interesting to note the SPR chips modified with only saccharide still revealed some adsorption of LDL onto their surface. When the surface 8366

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Protein Secondary Structure Analysis by Circular Dichroism (CD) Spectroscopy. Circular dichroism (CD) spectroscopy is a powerful method in structural biology that has been used to examine protein, polypeptide, and peptide structures since the 1960s19 and is especially effective to study the secondary structure of proteins interacting with molecules in solution; secondary structure changes cause changes in both the position and intensity of CD bands.22 To confirm the interactions between the immobilized saccharide and LDL, CD spectra of LDL in glucose (Glc) and galactose (Gal) solutions were examined and compared with the spectrum in PBS buffer (Figure 8). The CD curves of Figure 6. 1/Req vs 1/C plot for SPR measurement of sample (5−2). The relationship is y = 2.19 × 10−4 + 4.54 × 10−13x (R = 0.9993). The apparent affinity constant KA for LDL of the sample (5−2) was calculated from y-intercept and gradient to be 4.82 × 108 M−1.

Figure 8. CD spectra of LDL in different solutions: (red) LDL in PBS (pH 7.4); (green) LDL in 0.1 M Glc/PBS; (blue) LDL in 0.1 M Gal/ PBS. The concentrations of LDL were all maintained at 100 μg/mL. Solution backgrounds were subtracted from all CD curves. The curves of LDL in Glc and Gal solutions differed noticeably from the curve of LDL in buffer solution. (Glc: glucose; Gal: galactose.) Figure 7. LDL adsorption amounts of SPR chips with different surface ratios of glucosyl to sulfonic groups. On the x axis, the input ratios of SAM solutions are presented in brackets, and the XPS detected ratios of surface composition given below. The maximum LDL adsorption was around 3.68 ng/mm2, when the composition ratio of saccharide to alkanesulfonate was around 1.2 (prepared using an input ratio of 5−2). (1−0/-Glc: exclusively attached saccharide-moieties (glucose, Glc) as a control).

LDL in Glc and Gal solutions differed noticeably from the curve for LDL in buffer solution, which suggested interactions between saccharide molecules and LDL proteins that induced the distortion of LDL structure and thus deviations in the CD spectrum. The analysis of CD data was performed using the CDSSTR23 algorithm within the software CDPro (http://lamar.colostate. edu/∼sreeram/CDPro/main.html) (Table 3), and the reference set SDP48 (IBasis=7) was chosen for its optimum simulated results. The proportion of α-helix structure of LDL in glucose (Glc) solution decreased to 34.2% compared to 39.3% for LDL dissolved in buffer solution only, while the proportion of β-strand remained almost constant. The proportions of turn

proportion of sulfonic groups increased, (with a corresponding decrease of glucosyl groups), the LDL adsorption dropped. The maximum LDL adsorption was around 3.68 ng/mm2, when the composition ratio of saccharide to alkanesulfonate was around 1.2 (prepared using an input ratio of 5−2). These phenomena (the adsorption on the pure saccharide modified surface and the maximum adsorption found for sample (5−2)) could not be explained by just the electrostatic interaction between LDL and the sulfonic groups on the surface of the SPR chips. Therefore, the “multiple-interaction model” (Figure 1) is assumed to support nicely the mechanism of LDL adsorption, as additional interactions must have occurred between the LDL protein and immobilized saccharide, to explain these experimental results. Both interactions of saccharide and alkanesulfonate with LDL play important roles in the LDL adsorption process; the influences of the two groups were related to the surface proportions of glucosyl and sulfonic groups. Thus when the surface composition changed, each interaction strength changed accordingly, and the maximum LDL adsorption was found at a mixed surface composition.

Table 3. Secondary Structures of LDL Calculated from CD Spectra Using the CDSSTR Program with Reference Set SDP48 (IBasis=7)a sample LDL LDL in Glc LDL in Gal

H(r)%

H(d)%

S(r)%

S(d)%

Trn%

Unrd%

NRMSDb

21.1 17.2

18.2 17.0

16.1 17.1

167 167

13.0 16.4

15.6 15.8

0.076 0.093

17.4

17.2

16.6

166

16.1

16.6

0.093

a H(r) = regular α-helix; H(d) = distorted α-helix; S(r) = regular βstrand; S(d) = distorted β-strand; Trn = turns; Unrd = unordered. b The NRMSD is a fit parameter which is a measure of the difference between the experimental ellipticities and the ellipticities of the backcalculated spectra for the derived structure.

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Hypercholesterolemia by Extracorporeal Low-Density-Lipoprotein Immunoadsorption with Polyclonal Apolipoprotein-B Antibodies. Metabolism 1993, 42 (7), 888−894. (4) Gordon, B. R.; Kelsey, S. F.; Dau, P. C.; Gotto, A. M.; Graham, K.; Illingworth, D. R.; Isaacsohn, J.; Jones, P. H.; Leitman, S. F.; Saal, S. D.; Stein, E. A.; Stern, T. N.; Troendle, A.; Zwiener, R. J.; Grp, L. S. Long-term effects of low-density lipoprotein apheresis using an automated dextran sulfate cellulose adsorption system. Am. J. Cardiol. 1998, 81 (4), 407−411. (5) Ramunni, A.; Morrone, L. F.; Baldassarre, G.; Montagna, E.; Saracino, A.; Coratelli, R. Effectiveness of long-term heparin-induced extracorporeal LDL precipitation (HELP) in improving coronary calcifications. Int. J. Artif. Organs 2003, 26 (3), 252−255. (6) Klingel, R.; Mausfeld, P.; Fassbender, C.; Goehlen, B. Lipidfiltration - safe and effective methodology to perform lipidapheresis. Transfus. Apher. Sci. 2004, 30 (3), 245−254. (7) Bosch, T.; Gahr, S.; Belschner, U.; Schaefer, C.; Lennertz, A.; Rammo, J.; Grp, D. S. Direct adsorption of low-density lipoprotein by DALI-LDL-apheresis: Results of a prospective long-term multicenter follow-up covering 12 291 sessions. Ther. Apher. Dial 2006, 10 (3), 210−218. (8) Prassl, R.; Laggner, P. Molecular structure of low density lipoprotein: current status and future challenges. Eur. Biophys. J. Biophys. 2009, 38 (2), 145−158. (9) Jeon, H.; Blacklow, S. C. Structure and physiologic function of the low-density lipoprotein receptor. Annu. Rev. Biochem. 2005, 74, 535−562. (10) Cummings, R. D.; Kornfeld, S.; Schneider, W. J.; Hobgood, K. K.; Tolleshaug, H.; Brown, M. S.; Goldstein, J. L. Biosynthesis of NLinked and O-Linked Oligosaccharides of the Low-Density Lipoprotein Receptor. J. Biol. Chem. 1983, 258 (24), 5261−5273. (11) Groth, T.; Huang, X. J.; Guduru, D.; Xu, Z. K.; Vienken, J. Immobilization of heparin on polysulfone surface for selective adsorption of low-density lipoprotein (LDL). Acta Biomater. 2010, 6 (3), 1099−1106. (12) Li, J.; Huang, X.-J.; Ji, J.; Lan, P.; Vienken, J.; Groth, T.; Xu, Z.K. Covalent Heparin Modification of a Polysulfone Flat Sheet Membrane for Selective Removal of Low-Density Lipoproteins: A Simple and Versatile Method. Macromol. Biosci. 2011, 11 (9), 1218− 1226. (13) Huang, X.-J.; Guduru, D.; Xu, Z.-K.; Vienken, J.; Groth, T. Blood Compatibility and Permeability of Heparin-Modified Polysulfone as Potential Membrane for Simultaneous Hemodialysis and LDL Removal. Macromol. Biosci. 2011, 11 (1), 131−140. (14) Fass, D.; Blacklow, S.; Kim, P. S.; Berger, J. M. Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module. Nature 1997, 388 (6643), 691−693. (15) Rabbani, N.; Godfrey, L.; Xue, M.; Shaheen, F.; Geoffrion, M.; Milne, R.; Thornalley, P. J. Glycation of LDL by Methylglyoxal Increases Arterial Atherogenicity A Possible Contributor to Increased Risk of Cardiovascular Disease in Diabetes. Diabetes 2011, 60 (7), 1973−1980. (16) Hemphill, L. C. Familial hypercholesterolemia: current treatment options and patient selection for low-density lipoprotein apheresis. J. Clin. Lipidol. 2010, 4 (5), 346−349. (17) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Self-assembled monolayers of thiolates on metals as a form of nanotechnology. Chem Rev 2005, 105 (4), 1103−1169. (18) Green, R. J.; Frazier, R. A.; Shakesheff, K. M.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Surface plasmon resonance analysis of dynamic biological interactions with biomaterials. Biomaterials 2000, 21 (18), 1823−1835. (19) Wallace, B. A.; Whitmore, L. Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases. Biopolymers 2008, 89 (5), 392−400. (20) Ulman, A. Formation and structure of self-assembled monolayers. Chem. Rev. 1996, 96 (4), 1533−1554. (21) Zeng, X. Q.; Zhang, Y.; Luo, S. Z.; Tang, Y. J.; Yu, L.; Hou, K. Y.; Cheng, J. P.; Wang, P. G. Carbohydrate-protein interactions by

and unordered structures increased to 32.2% in the presence of Glc, compared to 28.6% in buffer solution. LDL in Gal solution showed similar changes to LDL in Glc. The proportion of αhelix structure for LDL in Gal solution decreased to 34.6% while the proportion of turn and unordered structures increased to 32.7%. Those decreases in α-helix structure and increases in turn and unordered structures indicated the destruction of highly ordered α-helix structure and protein conformational changes because of interactions between LDL and saccharides.24 This supports the “multiple-interaction model”, since even free saccharides in solution can interact with LDL protein, as elucidated from the CD results presented above.

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CONCLUSIONS The “multiple-interaction model” (as shown in Figure 1), where the saccharide components participate in the LDL adsorption process at biomaterial surfaces, is proposed here. The model is supported by SPR results which show that LDL can adsorb onto a surface that only contained saccharide groups. As the surface composition of saccharide and alkanesulfonate changed, a maximum appeared in the adsorption of LDL, which means the additional interactions occurred apart from electrostatic interactions. Further support for the model was observed with the help of CD investigations that prove a free saccharide−LDL interaction also in solution causing protein conformation changes. This proposed “multiple-interaction model” differs from earlier views in this field and may allow for a better understanding of the interaction between LDL and LDL-receptor, the relationship between diabetes and hyperlipidemia, or the design of more efficient and low cost LDL adsorbers in the future.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

* Supporting Information S

Two additional figures. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the Fundamental Research Funds for the Central Universities (Grant No. 2013QNA4049), the National Natural Science Foundation of China (Grant No. 21274126), National “Twelfth Five-Year” Plan for Science & Technology Support of China (No. 2012BAI08B01), Zhejiang Provincial Innovative Research Team (Grant No. 2009R50004), and the Open Foundation of Key Laboratory of Advanced Textile Materials and Manufacturing Technology (No. 2012001).

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REFERENCES

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dx.doi.org/10.1021/la401464a | Langmuir 2013, 29, 8363−8369