Dynamic Protein Adsorption onto Dendritic Polyglycerol Sulfate Self

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Dynamic Protein Adsorption onto Dendritic Polyglycerol Sulfate Self-Assembled Monolayers Daniel David Stöbener, Florian Paulus, Alexander Welle, Christof Wöll, and Rainer Haag Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00961 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 18, 2018

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Dynamic

Protein

Adsorption

onto

Dendritic

Polyglycerol Sulfate Self-Assembled Monolayers Daniel David Stöbener1, Florian Paulus1, Alexander Welle2, Christof Wöll*2, and Rainer Haag*1 1

Institute of Chemistry and Biochemistry, Freie Universitaet Berlin, 14195 Berlin, Germany

2

Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces (IFG), 76344

Eggenstein-Leopoldshafen, Germany

KEYWORDS Vroman effect, anionic surface coatings, protein rearrangement, ionic interactions

ABSTRACT Biomaterial surfaces which are in contact with blood are often prone to unspecific protein adsorption and the activation of the blood clotting cascade. Hence, such materials usually must be functionalized with low-fouling or anticoagulant polymer coatings to increase their performance and durability with respect to various applications, for example as implants or in biomedical devices. Many coatings are based on anionic polymers, such as heparin, and are known to have pronounced anticoagulant effects. To assess the ability of a surface to prevent biofouling and to get further insight into its underlying mechanism, studies of the protein adsorption on self-assembled monolayers (SAMs) are often used as a predictive tool. In this article, we synthesized thioctic acid-functionalized dendritic polyglycerol sulfate (dPGS), which is a well-known synthetic heparin mimetic, and immobilized it onto gold model surfaces. The

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anionic dPGS SAMs were characterized via contact angle measurements and ellipsometry and compared to their neutral dendritic polyglycerol (dPG) counterparts with respect to their single protein adsorption of the two most abundant blood proteins albumin (Alb) and fibrinogen (Fib). In addition, we used QCM-D and ToF-SIMS as complementary techniques to investigate the dynamic, mixed and sequential adsorption of Alb and Fib. Our results clearly demonstrate an incomplete Vroman effect and indicate the rearrangement of the adsorbed protein layers, which is presumably drive by ionic interactions between the two proteins and the anionic dPGS surface.

INTRODUCTION For many biomedical devices which are in contact with blood, the surface of the biomaterial is essential to avoid unspecific protein adsorption or blood activation.1-5 Most bioinert material surfaces6 are developed utilizing low fouling coatings based on neutral polyethylene glycol (PEG)7-10, negatively charged heparin-like polyanions11-15 or various zwitterionic polymers.16-18 In addition, self-assembled monolayers (SAMs) of functional polymers on gold have been intensively used as model surfaces to study unspecific and specific protein adsorption.9-10 In most studies, only single serum proteins, such as albumin (Alb), pepsin, lysozyme, and fibrinogen (Fib) were used.10,

19-20

In biomedical systems, however, the surface is exposed to a complex

mixture like blood or serum with more than 2500 individual proteins. In such situations, small and highly abundant proteins like Alb (55%, 65 kDa) will adsorb rapidly onto the biomaterial surface but will be slowly replaced by larger, less abundant proteins like Fib (7%, 340 kDa). This so-called Vroman effect21 occurs on a time-scale ranging from minutes to hours and describes the dynamic behaviour of the protein corona adsorbed on biomaterial surfaces and is therefore highly relevant for the fate of implants and nanoparticles in vitro and in vivo.


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We have demonstrated that dendritic polyglycerol (dPG) can efficiently protect material surfaces from unspecific protein adsorption and reduce the adhesion of undiluted blood serum proteins three times more efficiently than the gold standard PEG.20 In addition, due to its “tree-like” nature, dPG shields rough surfaces, such as poly(ether imide) (PEI) membranes, more efficiently from non-specific protein adsorption than linear PEG chains.22 Further, multivalent dendritic polyglycerol sulfates (dPGS) which efficiently inhibit inflammation in vivo and bind to various inflammatory factors have been introduced.23 As dPGS presents a highly active polymer therapeutic, the single protein interaction of lysozyme with dPGS of different molecular weights was investigated both experimentally and theoretically.24 To compare the blood protein interaction of the negatively charged dPGS to its neutral dPG counterpart, a more detailed study of the dynamic adsorption of important blood proteins on well-defined SAM-coated model surfaces is required. Herein, we describe the synthesis of thioctic acid functionalized dPGS and dPG and their SAMs on gold to investigate the dynamic and competitive protein adsorption by the complementary techniques Quartz Crystal Microgravimetry with Dissipation Monitoring (QCM-D) and Time-ofFlight Secondary Ion Mass Spectrometry (ToF-SIMS) to predict their performance as biomedical coatings. EXPERIMENTAL SECTION All materials and standard characterization methods used as well as detailed descriptions of the synthetic procedures are given in the Supporting Information. Gold surface modification and characterization. Cleaning Procedure for Gold-Coated QCM-D Sensors. QCM-D sensors were ozone-treated for 5 min in an UV/Ozone ProCleaner from BioForce Nanosciences Inc. (Ames, USA). Further chemical cleaning was accomplished by


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immersing the gold-coated sensors in a mixture of MQ water, ammonia, and hydrogen peroxide (ratio 5:1:1) at 80 °C for 5 min and rinsing with MQ and ethanol, followed by drying in a stream of nitrogen and a subsequent 5 min treatment in the UV/ozone oven. The cleaning of the gold surface was performed to remove adsorbed organic residues, which would have affected the selfassembly of the monolayer. Online Monitoring of the Coating Process by Quartz Crystal Microbalance Measurements. The gold surface modification was performed online in a QCM-D and offline outside of the QCM-D chamber to verify the transferability to other substrate geometries. In the online coating approach, the deposited, solvated polymer mass of thioctic acid-functionalized dPGS and dPG assembled on gold surfaces was determined by monitoring the change in resonance frequency (∆f) and dissipation (ΔD) of a piezoelectric quartz crystal over time. Changes in the fundamental frequency (4.95 MHz) and in overtones 3 to 13 were measured. For calculation of the layer thickness, the Sauerbrey model was chosen. Calculations were conducted using the software package QTools considering the third overtone. The fluid density (water) was set to 1000 kg m-3 and the layer density was estimated to be 1100 kg m-3. All solvents used during the measurements were degassed for 20 min in an ultrasonic bath. Phosphate buffered saline (PBS) tablets were dissolved in MQ (according to the manufacturers instruction), the solution was filtered (0.22 µm), and the pH was adjusted to 7.4 adding NaOH or HCl, respectively. Cleaned sensor chips were inserted into the flow chamber and equilibrated under water flow (0.1 mL min-1) until the baseline was constant. Polymer solutions in water (MQ, 0.1 mM) were flown into the chamber for 8 min to reach a concentration equilibrium inside the flow chamber. The flow was stopped, and SAM formation was monitored under static conditions for 3 hours. Subsequently, the surfaces were rinsed with water (0.1 mL min-1) until saturation was reached.


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The areal mass adsorbed and the thickness were calculated, and the coated chips were characterized via water contact angle measurements and ellipsometry. Offline coating of the gold substrates was performed by immersing the sensor chips into a polymer solution in water (MQ, 0.1 mM) for 3 hours. The sensors were thoroughly rinsed with water and characterized via contact angle measurements and ellipsometry. Protein adsorption by Quartz Crystal Microbalance Measurements. The polymer coated gold chips were inserted into the flow chamber and equilibrated under PBS flow (0.1 mL min-1) until the baseline was constant. Single protein adsorption of Alb and Fib was measured by flowing the respective protein solutions in PBS (1 mg mL-1) into the chamber for 25 min (0.1 mL min-1). Subsequently, the surfaces were rinsed with PBS (0.1 mL min-1) until saturation was reached. Mixed protein adsorption of Alb and Fib (8:1) was measured by flowing the protein solutions in PBS (1 mg mL-1) into the chamber for 25 min (0.1 mL min-1) and by leaving the protein mixture in the flow chamber at 0 mL min-1 to monitor structural changes of the adsorbed proteins on the coated surfaces. Subsequently, the surfaces were rinsed with PBS (0.1 mL min-1) until saturation was reached. Sequential protein adsorption of Alb and Fib (8:1) was measured by flowing Alb solutions in PBS (0.89 mg mL-1) into the chamber for 25 min (0.1 mL min-1), rinsing with PBS (0.1 mL min-1) until saturation was reached. Subsequently, a solution of Fib in PBS (0.11 mg mL-1) was flown over the coated surface for 25 min (0.1 mL min-1) and the sensor chip was rinsed with PBS (0.1 mL min-1) until saturation was reached. Contact Angle Measurements. The sessile drop method was applied to determine the static water contact angle on cleaned and polymer-coated gold surfaces at room temperature. Therefore, a drop of MQ (2 µL) was placed onto the respective surface and a photo was taken. Contact angles were determined with an ellipse-fitting model. For each substrate, contact angles


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were measured on three different spots to determine the homogeneity of the coating and at least three independent substrates (n = 3) were investigated to determine reproducibility. Mean contact angles of each substrate were averaged. Ellipsometry Measurements to determine Dry Thickness of the Coatings. Dry layer thicknesses of polymer coatings were determined by multiangle spectroscopic ellipsometry at 50°, 60° and 70°. Parameters of the cleaned gold-coated QCM-D sensors were determined and taken as fixed values for the following polymer layer thickness modeling. After online coating in the QCM-D flow chamber and offline coating, the layer thickness was measured at wavelengths from 370 to 1050 nm and fitted using a model consisting of the previously measured gold layer with fixed parameters. There was a Cauchy layer with fixed refractive index of n = 1.5 and air as the surrounding medium. ToF-SIMS. QCM-D sensor crystals coated with dPGS were rinsed after protein adsorption experiments with pure water to remove buffer salts and dried in nitrogen. ToF-SIMS (Time-ofFlight Secondary Ion Mass Spectrometry) was performed on a TOF.SIMS5 instrument (IONTOF GmbH, Münster, Germany). This spectrometer is equipped with a bismuth cluster primary ion source and a reflectron type time-of-flight analyzer. UHV base pressure was < 10-8 mbar. For high mass resolution, the Bi source was operated in the “high current bunched” mode providing short Bi3+ primary ion pulses at 25 keV energy and a lateral resolution of approx. 4 µm. The short pulse length of 1.2 ns allowed for high mass resolution. Primary ion doses were kept below 1011 ions/cm2 (static SIMS limit) for all measurements. Spectra were calibrated on the omnipresent C-, CH-, CH2-, C2-, C3-; or on the C+, CH+, CH2+, and CH3+ peaks. Based on these datasets chemical assignments were determined. 32 fragments characterizing amino acids25 were used for principal component analysis (PCA)26 using a GUI for MATLAB provided by Daniel


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Graham. 6 fields of view of 250×500 µm2 from each sample were analyzed and subjected to PCA. The datasets were normalized on the sum of peak areas and mean-centered. PCA creates linear combinations from the original 32-dimensional data set containing decreasing levels of variance. Each principal component contains loadings from all available signals (dimensions). PCA is totally unsupervised, i. e. independent of the origin of the datasets. This information is used after PCA to mark the scores and to provide confidence intervals shown in the scores plot. RESULTS AND DISCUSSION Polymer Synthesis and Functionalization The synthesis of dPGS-phthalimide yielded polymers with a molecular weight of 12 kDa and 4.5 phthalimide groups per molecule. After deprotection to the dPGS-amine, an average residue of 0.4 phthalimide groups per molecule which were not accessible to cleavage remained. The dPGS-amine was functionalized with thioctic acid (TA) via amide coupling using EDCl as a coupling reagent in a DMF/water (3:1) mixture. A degree of functionalization of 0.5 TA groups per dPGS molecule was achieved which was sufficient for the formation of SAMs on gold coated QCM-D sensors. The remaining amine groups accessible on the dPGS surface were quenched with ethyl isothiocyanate to eliminate remaining positive charges which could influence the protein adsorption behaviour. About 2 of the remaining amine groups could be converted into thiourea groups, leaving a negligible amount of 2 amine groups on each dPGS molecule.


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Scheme 1. Synthesis of dPGS-amine and dPGS-TA. (i) Glycidol/THF, NMP, 120 °C, 18h; (ii) 1/THF, NMP, 120 °C, o.n.; (iii) H2NSO3H, NMP, 90 °C, 48h; (iv) NaBH4, H2O, 50 °C, 15 h; (v) CH3COOH, H2O, 85 °C, 7 h, yield over four steps: 71%; (vi) Thioctic acid, EDCl, DMF/H2O, 50 °C, 4 h, rt., o.n., yield: 86%; (vii) EtNCS, DMF/PBS, rt., o.n., yield: 79%. SAM formation on Gold Substrates Coating of dPGS and dPG monolayers on gold was performed by online monitoring of the SAM formation using QCM-D and by offline incubation of QCM-D sensor chips. dPGS was coated from a 1 mM solution in aqueous NaCl (1 M) to screen the negative charges of the polymer and to increase the dPGS packing density on the gold surface, while dPG coatings were conducted from 1 mM solutions in water (MQ). The wetting behaviour and the thickness of the SAMs were characterized by contact angle and ellipsometry measurements, respectively (Table 1, column 2


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and 3). dPGS-functionalized surfaces showed high hydrophilicity with CAs in the immeasurable range below 10°, whereas compared to the cleaned, uncoated gold a slight decrease in CA was observed for dPG layers which is in line with earlier measurements of dPG-thioctic acid esters on gold.20 The water CAs and layer thicknesses of both types of monolayers determined after online monitoring as well as after offline coating were in good agreement and the results summarized in Table 1 represent averaged values. The lower average thickness of dPGS compared to dPG SAMs indicates a less dense packing of dPGS on the surface, which can be explained by the electrostatic repulsion of the negatively charged sulfate groups on the gold surfaces. However, CAs below 10° indicated a complete and homogeneous coverage of the gold surfaces with anionic dPGS layers. Table 1. Static water contact angle (CA), dry layer thickness (ddry), areal mass (ma) and wet layer thickness (dwet) of dPGS and dPG SAMs. Substrate

CA [°]

ddry [nm] a

ma [ng cm-2] b

dwet [nm] c

Au

40 ± 6 (n=7)

-

-

-

dPGS

< 10 (n=5)

1.4 ± 0.7 (n=4)

600 ± 120 (n=3)

5.4 ± 1.1 (n=3)

dPG

31.7 ± 4.2 (n=3)

3.2 ± 0.9 (n=3)

1250 ± 120 (n=3)

11.4 ± 1.1 (n=3)

a

determined via ellipsometry; b determined from QCM-D measurements via the Sauerbrey equation; c determined from QCM-D measurements using ma and an average layer density of 1100 kg m-3. Monitoring SAM formation by QCM-D online coating also confirmed a more efficient functionalization of the gold substrates by the more densely packed, neutral dPG. Using the Sauerbrey relation, a significantly higher areal mass and wet thickness was determined for the dPG layers (Table 1, entry 3 and 4). Representative frequency and dissipation curves of the QCM-D online coating experiments are shown in Figure 1. The slightly lower change in


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frequency Δf in case of dPGS (Figure 1a, blue curve) compared to dPG (Figure 1b, green curve) illustrates the different adsorption behaviour of the two polymers on the gold substrates. In addition, the slightly higher change in dissipation ΔD (Figure 1b, grey curve) further indicates a higher packing density of the dPG coatings.

Figure 1. Representative frequency and dissipation curves (3rd overtone) of the dPGS (a) and dPG (b) adsorption on gold sensor chips measured by QCM-D. Protein adsorption and coadsorption Single protein adsorption of the two important blood proteins Fib (7%) and Alb (55%) was performed on dPGS coated gold substrates as well as on dPG SAMs, known to have protein repellant properties, as controls. Figure 2 shows representative frequency and dissipation curves measured by QCM-D. Fib and Alb adsorption on dPGS were determined as 2020 ng cm-2 (Figure 2a, blue curve) and 310 ng cm-2 (Figure 2b, blue curve), respectively, which is in the range of a tightly packed monolayer in case of Fib27-28 and a more loosely packed Alb layer.29-31 Both proteins showed only minor adsorption on dPG SAMS with 7.3 ng cm-2 and 3.1 ng cm-2 in case of Fib and Alb, respectively. These results indicate the non-specific, electrostatic interactions between the positively charged domains of the two proteins with the negatively charged dPGS


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coated gold substrates. The affinity of serum proteins to interact with sulfated PGs via ionic interactions was recently investigated in solution using dPGS and dPGS coated gold nanoparticles.32 Compared to non-sulfated dPG, dPGS showed enhanced protein adsorption driven by ionic interactions and enhanced cellular uptake via the formed protein corona.32

Figure 2. Representative frequency and dissipation curves (3rd overtone) for the adsorption of Fib (a) and Alb (b) on dPGS (blue curves) and dPG (green curves) SAMs. To examine potential differences in the protein adsorption affinity, the adsorption of a mixture of Alb and Fib with a physiologically typical ratio of 8:1 and a sequential protein adsorption of Alb and Fib were investigated. The areal mass adsorbed on dPGS SAMs from the Alb/Fib (8:1) protein mixture was 750 ng cm-2 (Figure 3a), which indicates a mixed adsorption of Alb and Fib if compared to the values obtained from the respective adsorptions of the single proteins. The sequential adsorption of Alb and Fib revealed

similar results with 310 ng cm-2 after Alb

adsorption and 690 ng cm-2 after the subsequent Fib adsorption. These results either indicate an incomplete coverage of the dPGS SAMs by Alb and the consecutive coverage of still accessible dPGS areas by Fib, or the additional adsorption of Fib onto the Alb-covered dPGS surface.


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Figure 3. Representative frequency and dissipation curves for the adsorption of a physiological Alb/Fib (8:1, 1 mg mL-1) mixture (a) and for the sequential adsorption of Alb (0.89 mg mL-1) and Fib (0.11 mg mL-1) (b) on dPGS SAMs. To investigate the structure and composition of the adsorbed protein layers on the dPGS coated gold substrates, the plain and protein loaded dPGS coated sensor chips were analyzed via ToFSIMS. SIMS, being a rather destructive surface analysis, breaks down proteins by ion bombardment yielding fragments of the amino acid side groups that can be well-assigned to a chemistry and thereby to an individual amino acid.25, 33 Only CH4N+, being to only fragment of glycine is also found in other amino acids. To make use of a multi-dimensional dataset consisting of more than 30 signals, a principal component analysis was performed. Figure 4 shows the scores plot for the first two principal components of SIMS data of protein adsorbates, capturing 82% of the total variance, detected on the dPGS coated sensor crystals after the respective QCM-D experiments. As shown, both single protein adsorption data sets (blue and cyan) are well separated, distinguishable and homogeneous as concluded from their narrow distributions and confidence limits (95% confidence limits shown as ellipses). Further, it can be concluded that protein adsorption from a physiological BSA/Fib (8:1) mixture (pink crosses) clearly shows the formation of another, less homogeneous, protein adsorbate. Especially on principal component 1,


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plotted on x, scores are found that do not resemble either a pure albumin or a pure fibrinogen layer, nor a random protein mixture. The high score in PC1 rather indicates structural changes in the adsorbed proteins. These changes lead to a new amino acid pattern exposed to the sampling (depth) range of the SIMS experiment. If the dPGS surface is exposed to both proteins sequentially (first BSA, then followed by Fib according to Fig. 3b) another adsorbate is formed. Neither does the pre-adsorbed albumin passivate the surface against further fibrinogen adsorption, nor does the fibrinogen fully replace the albumin. Hence, we conclude that an incomplete Vroman effect is observed and that partial rearrangement of the protein structures are possible.

Figure 4. ToF-SIMS scores of principal components 1 and 2 of dPGS coatings loaded with proteins from QCM-D experiments with pure Fib (blue), pure Alb (cyan), mixed (pink crosses) and sequentially adsorbed Alb and Fib (red stars). 95% confidence intervals are shown.


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Figure 5. Amino acid loadings of principal components 1 (a), (44: C2H6N, Alanine; 70: C3H4NO, Asparagine, 73: C2H7N3, Arginine, 84: C5H10N, Lysine; 130: C9H8N, Tryptophan) and 2 (b), (45: CHS, Cysteine; 110: C5H8N3, Histidine). SUMMARY AND CONCLUSIONS To study the single, competitive and dynamic protein adsorption of the two blood proteins Fib and Alb on anionic dPGS surfaces, we used the thioctic acid-functionalized dPGS to form stable SAMs on gold model substrates. We developed a highly scalable synthetic protocol to obtain dPGS-TA and investigated the SAMs by online monitoring via QCM-D, characterization via ellipsometry and water CA measurements. Compared to the charge neutral dPG, dPGS monolayers were less densely packed on the gold substrates, which can be explained by the repulsion of the highly abundant negatively charged sulfate groups decorating the dPGS surface. However, layer thicknesses and water CA data indicated a homogeneous distribution of dPGS molecules on the surfaces. QCM-D adsorption experiments of the single proteins Fib and Alb revealed the formation of densely packed protein layers in case of Fib and a more loosely packed protein layer in case of Alb. As no significant protein adsorption was detected on the neutral dPG control surfaces, our results indicate a non-specific, electrostatic interactions between the positively charged domains of the two proteins with the negatively charged dPGS coated gold


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substrates. The complementary study of the mixed and sequential Alb/Fib adsorption via QCMD and ToF-SIMS revealed, that in both cases, the conformations of the adsorbed protein layers differ from each other as well as from those of the single proteins. Moreover, only an incomplete Vroman effect was observed, since Alb neither passivates the surface from further Fib adsorption, nor does Fib completely replace the pre-adsorbed Alb layer. Hence, we conclude that the partial rearrangement of the protein structures is possible, which is presumably driven by ionic interactions between the proteins and the anionic dPGS monolayers. AUTHOR INFORMATION Corresponding Authors *R.H.: Tel: +49-30-838-52633. E-mail: [email protected] *C.W.: Tel: +49-721-608-2-3934. E-mail: [email protected] ORCID Rainer Haag: 0000-0003-3840-162X Christoph Wöll: 0000-0003-1078-3304 Alexander Welle: 0000-0002-3454-6509 Author Contributions Daniel David Stöbener and Florian Paulus performed the synthesis and characterized the compounds. Alexander Welle performed the ToF-SIMS measurements and data analysis. Christof Wöll and Rainer Haag conceived the concept. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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Funding Sources Helmholtz portfolio project “multimodal imaging” and German Science Foundation SFB 765. ACKNOWLEDGMENT The authors would like to thank Daniel Graham, University of Washington, for developing the PCA data analysis toolbox used in this study, funded via NIH grant EB-002027. We thank Michael Zierke and Dr. Marc Behl for helpful discussions in the dPGS-Amine synthesis. Dr. Wiebke Fischer is acknowledged for her support in finalizing this manuscript. We further thank Dr. Lei-Xiao Yu for conducting additional QCM-D measurements. SUPPORTING INFORMATION Materials and characterization methods used, details on polymer synthesis and characterization REFERENCES (1) Ratner, B. D. The Catastrophe Revisited: Blood Compatibility in the 21st Century. Biomaterials 2007, 28, 5144-5147. (2) Chen, S.; Jiang, S. An New Avenue to Nonfouling Materials. Adv. Mater. 2008, 20, 335338. (3) Ladd, J.; Zhang, Z.; Chen, S.; Hower, J. C.; Jiang, S. Zwitterionic Polymers Exhibiting High Resistance to Nonspecific Protein Adsorption from Human Serum and Plasma. Biomacromolecules 2008, 9, 1357-1361. (4) Zhang, Z.; Zhang, M.; Chen, S.; Horbett, T. A.; Ratner, B. D.; Jiang, S. Blood Compatibility of Surfaces with Superlow Protein Adsorption. Biomaterials 2008, 29, 4285-4291. (5) Blaszykowski, C.; Sheikh, S.; Thompson, M. Surface Chemistry to Minimize Fouling from Blood-Based Fluids. Chem. Soc. Rev. 2012, 41, 5599-5612. (6) Wei, Q.; Becherer, T.; Angioletti-Uberti, S.; Dzubiella, J.; Wischke, C.; Neffe, A. T.; Lendlein, A.; Ballauff, M.; Haag, R. Protein Interactions with Polymer Coatings and Biomaterials. Angew. Chem., Int. Ed. 2014, 53, 8004-8031. (7) Lee, J. H.; Lee, H. B.; Andrade, J. D. Blood Compatibility of Polyethylene Oxide Surfaces. Prog. Polym. Sci. 1995, 20, 1043-1079. (8) Arima, Y.; Toda, M.; Iwata, H. Complement Activation on Surfaces Modified with Ethylene Glycol Units. Biomaterials 2008, 29, 551-560. (9) Prime, K.; Whitesides, G. Self-Assembled Organic Monolayers: Model Systems for Studying Adsorption of Proteins at Surfaces. Science 1991, 252, 1164-1167.


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Dynamic Protein Adsorption onto Dendritic Polyglycerol Sulfate Self

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