Impact of Bioactive Peptide Motifs on Molecular Structure, Charging

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Impact of bioactive peptide motifs on molecular structure, charging and non-fouling properties of poly(ethylene oxide) brushes Ognen Pop-Georgievski, Ralf Zimmermann, Ilya Kotelnikov, Vladimir Proks, Dirk Romeis, Jan Kucka, Anja Caspari, Frantisek Rypacek, and Carsten Werner Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00441 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Impact of bioactive peptide motifs on molecular

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structure, charging and non-fouling properties of poly(ethylene oxide) brushes

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Ognen Pop-Georgievski, †, ‡,*Ralf Zimmermann, ǁ, ‡,* Ilya Kotelnikov, † Vladimir Proks, †

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Dirk Romeis, ǁ Jan Kučka, † Anja Caspari, ǁFrantišek Rypáček † and Carsten Werner ǁ , §

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†

Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovskeho nam. 2,

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162 06 Prague 6, Czech Republic. ǁ

8 9 10

Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials Dresden, Hohe Str. 6, 01069 Dresden, Germany.

§

Technische Universität Dresden, Center of Regenerative Therapies Dresden, Tatzberg 47,

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01167 Dresden, Germany

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KEYWORDS: biomimetic surface engineering, polydopamine, poly(ethylene oxide), electro-

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kinetics, streaming current measurements, click chemistry, peptide radiolabeling, RGD

ACS Paragon Plus Environment

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ABSTRACT

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Polymer layers capable of suppressing protein adsorption from biological media while

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presenting extracellular matrix derived peptide motifs offer valuable new options for

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biomimetic surface engineering. Herein, we provide detailed insights into physico-chemical

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changes induced in a non-fouling poly(ethylene oxide) (PEO) brush/polydopamine (PDA)

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system by incorporation of adhesion ligand (RGD) peptides. Brushes with high surface chain

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densities (σ ≥ 0.5 chains·nm-2) and pronounced hydrophilicity (water contact angles ≤ 10°)

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were prepared by end-tethering of hetero-bifunctional PEOs (Mn ~ 20000 g·mol-1) to PDA-

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modified surfaces from a reactive melt. Using alkyne distal end-group on the PEO chains,

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azidopentanoic-bearing peptides were coupled through a copper-catalyzed Huisgen azide-

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alkyne „click‟ cycloaddition reaction. The RGD surface concentration was tuned from

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complete saturation of the PEO surface with peptides (1.7×105 fmol·cm-2) to values which

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may induce distinct differences in cell adhesion ( H3O+) from the electrolyte . Despite

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the distinct differences observed in the electrokinetic analysis of the surfaces bearing different

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amounts of RGD, it was found that the peptide presence on PEO(20000)-PDA layers does not

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have a significant effect on the non-fouling properties of the system. Notably, the presented

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PEO(20000)-PDA layers bearing RGD peptides in the surface concentration range 5.9–


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1.7×105 fmol·cm-2 reduced the protein adsorption from fetal bovine serum to less than

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30 ng·cm-2, i.e. values comparable to the ones obtained for pristine PEO(20000)-PDA layers.

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INTRODUCTION

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The understanding of the microenvironmental cues of the extracellular matrix (ECM) is

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decisive for the successful development of cell therapies, tissue repair procedures and

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implants.1 Approaches to investigate the behavior of cells on artificial materials include

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surface functional group modifications,2 micro-patterns of fibronectin

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polymers4 and ECM proteins5, variation of stiffness and nanotopology6-8, etc. Arguably, one

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of the most important challenges for gaining control of cell behavior is the design of patterned

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surfaces which mimic the composition of the ECM and/or simulate the cell−cell interactions

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on micro- and nano-level.9 One of the main strategies in surface engineering relies on

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developing libraries of biomimetic peptides in order to produce tissue compatible materials.

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RGD, a fibronectin-derived peptide sequence, has been used as the golden standard for the

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binding of integrin receptors expressed by various cell types.10 Statistical immobilization of

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RGD peptides was performed on model gold surfaces bearing oligo(ethylene oxide) (OEO)

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self-assembled monolayers (SAMs) through a maleimide distal-end functionality.11-12 The

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OEO moiety has been used to provide specific ligand–receptor interactions by eliciting the

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RGD sequences. It has been supposed that the presence of the short OEO chains decreases

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nonspecific adsorption of proteins from the cultivation media, as in the case of pristine OEO

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SAMs.11-12 Densely packed PEO polymer chains comprising of 50—500 EO monomer units

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are well known to provide higher steric hindrance and thus better protein repellent properties

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than the 1—6 EO units of OEO SAMs. The adsorption of polyionic PEO-grafted copolymers

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such as poly(l-lysine)-g-poly(ethylene oxide) PLL-g-PEO has been demonstrated to result in

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non-fouling layers on the surfaces of negatively charged metal oxides such as TiO2, Ta2O5,

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Nb2O5 and SiO2.13-16 This approach was used for the formation of PLL-g-PEO copolymer


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and -arrays of

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layers bearing about 30 and 70 fmol·cm-2 of RGD and its scrambled equivalent RDG peptides,

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respectively.14-15 An even more universal surface modification platform can be obtained by

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using a polydopamine (PDA) anchor layer. It has been shown that this bioinspired

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multifunctional coating can be prepared on a wide range of inorganic and organic materials,

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including noble metals, oxides, polymers, semiconductors, and ceramics.17-19 The unsaturated

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indole rings and catechol groups of the various monomer units constituting PDA20 can be

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used for secondary reactions to prepare non-fouling polymer brushes by "grafting to-"19, 21 and

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"grafting from-"22 methods. Recently, our group has reported the "click & seed" approach for

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the biomimetic modification of solid surfaces.23 The proposed approach is based on the

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creation of a non-fouling PEO brush on a polydopamine anchoring layer19, 21 and its capacity

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for selective follow-up modifications with various ligands via a copper-catalyzed Huisgen

12

azide-alkyne ‘‘click’’ cycloaddition reaction (CuAAC). Thus, the desired surface

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concentration of ligands can be precisely controlled by adjusting the peptide concentration in

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the reaction mixture.23

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However, the changes in the physicochemical properties of the polymer films as well as the

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changes in resistance to non-specific protein adsorption due to the immobilization of peptide

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sequences remained to be addressed. Recent advances in label free diagnostics have

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demonstrated the importance of functional polymer coatings providing high biorecognition

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element immobilization (BRE) capacity while simultaneously exhibiting fouling resistance

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from complex media.24 While the preservation of the non-fouling properties upon

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immobilization of BRE’s has been a central issue in sensing applications, it has been

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neglected in the case of tissue engineering applications. The immobilization of neutrally

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charged RGD peptide sequence at surface densities ranging from 0.7 to 1.7×104 fmol·cm-2,

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has been expected to maintain the originally observed non-fouling properties of layers

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unaffected.11-12 The in-situ null ellipsometry study of Textor et al.15 has critically addressed


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the changes in the fouling behavior induced by the presence of RGD and RDG peptides at

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surface concentrations of about 30 fmol·cm-2, but did not address the non-fouling behavior of

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the layers in large surface concentration window. The changes in the physico-chemical

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properties of the surface by the immobilization of biomimetic ligands, and the consequent

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changes in resistance to fouling can be detrimental for the actual surface engineering

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application that these surfaces are intended for. Therefore, the main aims of this study were

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(i) to probe the screening of the PDA layer charge by the dense attachment of PEO chains; (ii)

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to analyze the physico-chemical changes induced to non-fouling PEO-PDA surfaces by the

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immobilization of bio-adhesive RGD peptide ligands with surface concentration of 5.9 to

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1.7×105 fmol·cm-2 (corresponding to hexagonally packed ligands with distances of 180.8 to

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1.1 nm, respectively) and (iii) to assess the non-specific adsorption of globular proteins to

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these surfaces. To address these challenges, the end-tethering of dense brushes on PDA-

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modified substrates were performed from reactive polymer melt of heterobifunctional PEOs,

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α-aminoethyl-ω-methoxy-PEO and α-aminoethyl-ω-alkyne-PEO of 20000 g·mol-1. The

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presence of the alkyne functionality at the chain distal-ends of PEO(20000) was used for the

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immobilization of RGD peptides in surface concentrations ranging from complete saturation

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of the surface, providing one specifically bound RGD sequence per PEO chain, to values

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below 600 fmol·cm-2 which were found to be significant for monitoring integrin-mediated cell

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attachment, migration and spreading, focal adhesion dynamics and adhesion strength.10, 25-26

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The actual RGD surface concentration was precisely tuned by using

21

derivate and monitoring the increase in samples radioactivity using a radioactivity assay. All

22

layers were characterized in terms of their thicknesses, hydrophilicity and chemical

23

composition utilizing spectroscopic ellipsometry (SE), water contact angle (CA) goniometry,

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infrared reflection-absorption spectroscopy (IRRAS) and X-ray photoelectron spectroscopy

25

(XPS) , respectively. The screening of the PDA anchor layer charge by the immobilization of


125

I-radiolabeled RGD

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PEO chains was probed by streaming current measurements using the Microslit Electrokinetic

2

Set-up (MES).27 Brush profiles have been calculated on the basis of the self-consistent field

3

method28 for the detailed analysis of the electrohydrodynamics at the brush-solution interface.

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The MES was further used to characterize the changes in the interfacial charging introduced

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to alkyne-PEO(20000)-PDA layers by the immobilization of RGD peptides. The observed

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trends were modelled considering different scenarios for the interfacial charge formation at

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the RGD functionalized brushes. The adsorption from single protein solutions and from

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multicomponent, serum-containing media to pristine and RGD modified surfaces was

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monitored in situ via surface plasmon resonance (SPR).

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With all this we were able to analyze and interpret changes in the physicochemical and non-

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fouling properties of PDA-anchored PEO layers upon immobilization of model biomimetic

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peptide sequences such as RGD. The herein proposed surface analysis methodology targets

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the non-fouling properties of biomimetic surfaces incorporating peptide adhesion motifs and

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polymer brushes. However, the presented analysis can be adapted and implemented for

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exploring biomimetic surface engineering avenues aiming at different functionalities.

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EXPERIMENTAL SECTION

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All materials, chemicals, and reagents were used as received unless stated otherwise. The

18

detailed synthetic procedures, characterization methods, and further supporting analysis are

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provided in the Supporting Information (SI). In the following, a short summary of the sample

20

preparation and the characterization methods is presented.

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Grafting of PEO. Hetero-bifunctional PEOs (Rapp Polymere, Germany), α-aminoethyl-ω-

22

methoxy-PEO with number average molecular weights 22400 g·mol-1, and α-aminoethyl-ω-

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alkyne-PEO with a number average molecular weight of 17300 g·mol-1, were used for the

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preparation of the polymer brushes. The dispersity (Đ) of all polymers was 1.03 as stated by

25

the supplier. Although throughout the text we use the nominal values of ~20000 g·mol-1, all


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calculations were performed using the previously stated Mn’s. The PEO chains were grafted

2

from melt at 110 °C19 to substrates bearing stabilized PDA anchor layer (Scheme 1A).

3

Peptide

synthesis,

radio-labeling

and

immobilization.

The

azidopentanoyl–

4

GGGRGDSGGGY–NH2 peptide was synthesized on TentaGel R Rink Amide resin. The

5

purity of synthesized peptide (>98 %) was determined by high-performance liquid

6

chromatography on a gradient Knauer system with diode array detection (Phenomenex C18,

7

5-mm column, gradient elution with the following solvents: A, H2O with 0.1% TFA; B,

8

CH3CN with 0.1% TFA.) The molecular weight of the peptide was confirmed by matrix-

9

assisted

laser

desorption/ionization

time-of-flight

(MALDI-ToF)

on

a

Bruker

10

Biflex III mass spectrometer in reflector mode using 2,5-dihydroxy benzoic acid as a matrix

11

in a 5:1 matrix/sample ratio. The chloramine T/ascorbic acid radiolabeling method was

12

adapted for the conditions of the solid–phase peptide

13

reports

14

GGGRGDSGGGY(125I)–NH2 peptide on SiO2/Si control samples bearing alkyne-

15

PEO(20000)-PDA layers was performed utilizing a copper-catalyzed Huisgen azide-alkyne

16

‘‘click’’ cycloaddition reaction (CuAAC) from 5.8 × 10-8, 5.7 × 10-9 and 7.3 × 10-10 mol·mL-1

17

water solutions (Scheme 1B). The immobilization efficiency and percentage of non-bound

18

peptides was evaluated on the basis of radioassay analysis (Refer to SI for further

19

experimental details). The same immobilization procedure was followed for the binding of

20

non-labeled azidopentanoyl–GGGRGDSGGGY–NH2 in the surface concentration of 5.9 to

21

1.7×105 fmol·cm-2 on alkyne-PEO(20000)-PDA on SPR and EK substrates. The surface

22

related parameters of non-labeled peptides, i.e. surface density σpeptide and average distance

23

between supposedly hexagonally packed molecules =

24

accordance to the radioactivity findings (Table 1 and SI).

of

Proks

et

al.23

The

125

Iodine radiolabeling according the

immobilization


of

labeled

√

azidopentanoic–

were calculated in

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Scheme 1. (A) End-tethering of α-aminoethyl-ω-methoxy-PEO and α-aminoethyl-ω-alkyne-

2

PEO from reactive melt to substrates bearing PDA anchor layer. (B) Binding of RGD

3

peptides to alkyne-PEO(20000)-PDA layers utilizing a copper-catalyzed Huisgen azide-

4

alkyne ‘‘click’’ cycloaddition reaction.

5

6 7 8


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Table 1. Surface concentration, surface density and average distance between supposedly

2

hexagonally packed peptide molecules (D). Surface concentration

[fmol·cm-2]

[peptide·nm-2]

[nm]

1.7×105

1.0

1.1

3.0×103

1.9×10-2

7.9

5.7×102

3.4×10-3

18.4

30.9

1.9×10-4

78.7

5.9

3.5×10-5

180.8

3 4


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DATA ANALYSIS

2

Grafting Density, Distance Between Grafting Sites, and Structural State. The PEO

3

thicknesses obtained from the ellipsometry measurements were used to calculate the grafting

4

density =

5

=

6

ellipsometry,29-31 ρ = 1.09 g·cm-3 is the PEO bulk density, and NA is the Avogadro constant.

7

The ratio of the distance between PEO chains to the radius of gyration Rg

8

describes the overlapping of the tethered chains. Values of

9

polymer chains are in a "brush" state. Rg of PEO in water was calculated according to

√

and the distance between grafting sites supposing hexagonal packing

, where h is the layer thickness in the dry state as determined by

!"

PEO,

i.e.

!"

,

< 0.5 indicate that the

10

Kawaguchi et al.32

11

Brush profile. The thickness determined by ellipsometry according to a Cauchy-layer model

12

(Refer to SI) represents the extension of a the bushes under the assumption of an averaged

13

segment distribution in the direction normal to the surface.33 By contrast, real brushes show a

14

gradually decreasing polymer density with increasing distance from the supporting surface.34-

15

35

16

Đ is greater than 1, the segment distribution decreases more gradual in the outermost brush

17

region.28 As this region determines the electrohydrodynamics at the interface,36 we considered

18

dispersity effects in the evaluation of the streaming current data. For that purpose we

19

calculated the normalized brush profile for Đ = 1.03 on the basis of the self-consistent field

20

method developed by Milner et al.28 following the procedure reported in details elsewhere.37

21

For the analysis of the electrohydrodynamics at the interface the normalized profile was

22

converted in the real segment densities using an excluded volume parameter, ν, of 0.08 nm³

23

37

In the ideal case of a monodisperse polymer, brushes show a parabolic density profile.34 If

, a segment length, b, of 0.37 nm38 as well as the grafting densities, σ, and the average


10

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number of monomer units per polymer chain, Nav, given in (Table 2) following the procedure

2

reported in Ref. [37].

3

Streaming current measurements and data interpretation. Streaming current (Istr)

4

measurements were performed at varying pressures (∆P) across rectangular streaming

5

channels (length Lo = 20 mm, width ℓ = 10 mm, height H = 30 µm) formed by two brush-

6

coated sample surfaces using the Microslit Electrokinetic Set-up.27 Measurements performed

7

as a function of solution pH were started in the alkaline pH range. For each composition of

8

the electrolyte the samples were equilibrated for about 40 min prior to measurement.

9

The streaming data measured for the PEO brushes were analyzed applying the theory for

10

the electrohydrodynamics of diffuse soft interfaces considering all ion species (K+, H3O+, OH-

11

, and Cl-) present in the electrote.36 In short, the streaming current, Istr, caused by the transport

12

of mobile charges at the PDA/PEO/solution interphase depends on the hydrodynamic flow

13

field, V(X), and the distribution of mobile ions in the interfacial region. The hydrodynamic

14

flow field was calculated according to the generalized Brinkman equation.37 The distribution

15

of counter ions that compensate the charge of the PDA layer and RGD functionalized PEO

16

brushes was derived from the non-linear Poisson-Boltzmann equation.36 The streaming

17

current, Istr, resulting from the convective transport of counter ions is then given by:36

18 F

()*+ 2ℓ/0 4 5 678 9 :; 0.5 chains·nm-2) on the PDA modified surfaces is corroborated by the decrease of the

13

water CA from 69 ± 1° for the pristine PDA films to less than 10° for the PEO bearing one.

14

The presence of the more hydrophobic alkyne-group instead of the methoxy-group at the

15

distal-end of the PEO(20000) chains did not affect the hydrophilicity of the layers.

!"

16

The covalent attachment of the PEO layers to the PDA anchors has been probed by

17

complementary IRRAS and XPS analysis (Figure 1). The appearance of molecular signatures

18

for the C–O–C stretching mode at 1120 and 1149 cm-1 in the IRRAS spectra and the

19

predominance of C–O contributions at about 286.6 eV in the XPS spectra upon the grafting

20

prove the attachment of PEO chains to the PDA surface. Detailed analysis can be found in the

21

SI Complementary Results on Covalent Structure of PEO-PDA layers.

22 23


12

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Table 2. Average number of monomer units per polymer chain (Nav), ellipsometric thickness

2

(h), PEO grafting density (σ), distance between grafting sites supposing hexagonal packing of

3

the polymer chains (D), chain overlapping parameter (DPEO/2Rg PEO) and static water contact

4

angle (θ static).

Nav

Layers

PDAb

h

σPEO

DPEO

[nm]

[chains·nm-2]

[nm]

DPEO/2Rg PEO a

θ static [°]

13.1 ± 2.6

69 ± 1

PEO(20000)-OCH3

507

15.6 ± 2.8

0.5 ± 0.1

1.6 ± 0.1

0.1

7±1

PEO(20000)-alkyne

392

17.9 ± 3.1

0.7 ± 0.1

1.3 ± 0.1

0.1

7±1

5

a

6 7

b

Values of

!"

< 0.5 indicate that the polymer chains are in a "brush" state.

The PDA anchor layers used for grafting of PEO were of same thickness as the pristine PDA film.


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Figure 1. IRRAS (A) and high resolution C 1s XPS spectra (B) of PDA anchor (a), CH3OPEO(20000) (b) and alkyne-PEO(20000) (c)

layers. The insert in the IRRAS graph

represents the contributions in the region of C≡C stretching absorption. Measured spectra are presented with open circles, while their corresponding fitted envelopes are presented with lines. The individual contributions of different functional groups are represented with dashed lines.


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Brush profile and chain end distribution. The obtained molecular parameters for the PEO polymer brushes (Table 2) have been used to calculate the normalized density profile and the chain end distribution (Figure 2) utilizing the self-consistent field method. In the ideal case of monodisperse polymer chains (Đ = 1.0) the brush shows the characteristic parabolic profile. The dispersity of the PEO used in this study (Đ = 1.03) results in a deviation from the parabolic profile with a more gradual decrease of the segment density from the substrate in the direction to the PEO/solution interface. The chain end density increases from the supporting surface towards the middle of the brush. After reaching the maximum at a distance of 0.54 (in rescaled units), the chain end density decreases towards the solution phase with a more distinctive tail region for the disperse brush. For the analysis of the data from the electrokinetic measurements the normalized profiles were converted into real segment densities and chain end distributions (SI, Figure S5) using 0.08 nm3 for the excluded volume parameter, ν, 0.37 nm for the segment length, b, as well as the numbers given for the monomers per polymer chain and grafting densities in Table 2.


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Figure 2. Density profile (A) and chain end distribution (B) for PEO brushes with a dispersity Đ = 1.03 and monodisperse brushes (Đ = 1) in rescaled units. In line with the conditions of the electrokinetic experiments, the temperature was set to 295.15 K. For further details concerning profile calculation refer to text and Ref. [28]. The spatial coordinate x corresponds to the direction normal to the brush/solution interphase.


16

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Figure 3. Streaming current over applied pressure, Istr/∆P, as a function of pH in 1 mM KCl solution for the bare PDA layer (black) as well as the PDA layer after immobilization of CH3O-PEO(20000) (red) and alkyne-PEO(20000) (blue) brushes. Experimental data are represented as symbols and the solid lines represent simulation results. The simulation curves were obtained by adjustment of the hydrodynamic penetration length, 1/λo, according to the least

square

method:

CH3O-PEO(20000):

1/λo

=

22.1 nm,

alkyne-PEO(20000):

1/λo = 18.3 nm. The experimental data measured for the bare PDA-coated substrate were reproduced using 1/λo → ∞, corresponding to the absence of hydrodynamic screening by the brushes. The grafting densities and the number of repeat units per polymer chain were used as given in Table 2. Other parameters: Excluded volume: ν = 0.08 nm³, segment length: b = 0.37 nm, channel length: Lo = 20 mm, channel width, ℓ = 10 mm, channel height: H = 30 µm, viscosity of electrolyte: η = 0.954 mPa·s-1 and relative dielectric permittivity of the medium: εr = 79.5.


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Page 18 of 36

1

Streaming current data measured in 1 mM KCl solution as function of the electrolyte pH

2

are presented in Figure 3. The pristine PDA films show a point of zero streaming current

3

(PZSC) at about pH 4.9 above which the layer is negatively charged. The magnitude of the

4

negative streaming current increases with the degree of de-protonation of the PDA's carboxyl

5

and catechol groups at increasing pH values. After the immobilization of the PEO we

6

determined a similar PZSC for CH3O-PEO(20000) and alkyne-PEO(20000), indicating that

7

the charging of the PDA is not significantly affected by the grafting of the PEO. The excess

8

charge of the PDA layer is compensated by counter ions in the diffuse layer. The significant

9

drop of Istr/∆P after the immobilization of the PEO is the result of the hydrodynamic

10

screening of the counter charges by the immobilized PEO chains.37 The degree of

11

hydrodynamic screening depends on the extension of the brushes as compared to the

12

extension of the diffuse layer (∼9.7 nm in 1 mM KCl) and the segment density which

13

determines the penetration of the tangential flow into the brush.36

14

In order to reconstruct the experimental data shown in Figure 3 by the theory outlined

15

above, we converted the streaming current data of the PDA surface into zeta potentials using

16

Helmholtz-Smoluchowski equation39 thus providing the surface potential of the PDA surface

17

and counter ion distribution as function of the pH. Assuming that the interfacial charge

18

formation is not altered by the presence of the PEO brushes, the hydrodynamic penetration

19

length, 1/λo, was fitted according to the least-squares method to recover the experimental

20

data. Complete recovery of the experimental data was achieved with 1/λo = 22.1 nm for

21

CH3O-PEO(20000) brushes and 1/λo = 18.3 nm for alkyne-PEO(20000) brushes. As

22

electrokinetics has been proven to be very sensitive in the analysis of the charge and structure

23

of soft interfaces,36-37 the correlated evaluation of segment density distribution in swollen

24

PEO brushes enables the effective determination of thickness of swollen PEO brushes. This

25

enabled circumventing the main problems of in situ SE analysis for the determination of


18

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Langmuir

1

thickness of swollen films, i.e. strong correlation of thickness and optical parameters of the

2

swollen layer, as well as the minor optical contrast between the swollen brush and the

3

medium at measured wavelength range. The determined extension of the brushes is about

4

80 nm for CH3O-PEO(20000) and 69 nm for alkyne-PEO(20000), i.e. the hydrodynamic flow

5

field penetrates the brushes to less than 30% of their maximum extension in the direction

6

normal to the supporting surface (SI, Figure S5). Notably, the determined swollen brush

7

thicknesses are well below the theoretical end-to-end distance of fully stretched CH3O-

8

PEO(20000) and alkyne-PEO(20000) chains in all ttt conformation of 184 nm and 143 nm,

9

respectively, thus leaving high conformational freedom of the end-tethered chains.

10

The effective screening of the charged and highly fouling PDA anchor layer was further

11

demonstrated by SPR (incubation time of 15 min at constant flow rate of 20 µL·min-1)

12

utilizing different single protein solutions (human serum albumin (HSA), fibrinogen (Fbg),

13

and lysozyme (Lys)) and fetal bovine serum (FBS) (Table 3). The selected proteins span a

14

wide range of molecular weights, sizes, isoelectric points and structural stability. At

15

physiological pH, the structurally stable “hard” protein Lys (Mw = 14 kDa, 40 × 30 × 30 Å3,

16

isoelectric point pI = 11.4) carries a positive net charge, while the structurally unstable “soft”

17

proteins HSA (Mw = 67 kDa, spherical shape 72Å, pI = 4.7) and Fbg (Mw = 340 kDa, 450 ×

18

90 × 90 Å3, pI = 5.5) carry a negative net charge.21, 40-41 At the same time the adsorption from

19

FBS, one of the most frequently used complex biological media for in vitro cell culturing,

20

would emulate the behavior of the neat brushes in a cell seeding environment.

21

The adsorption studies on bare PDA films revealed deposits of 120 ± 30, 290 ± 60,

22

110 ± 30 and 270 ± 40 ng·cm-2 of adsorbed HSA, Fbg, Lys and FBS, respectively. These

23

values were close to a monolayer of proteins on bare gold indicating that the PDA surface

24

was fully covered, thus its properties were totally modified. The PEO polymer brushes

25

anchored to PDA reduced the protein adsorption from the main plasma proteins HSA, and


19

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Page 20 of 36

1

Fbg, and the structurally stable and positively charged Lys close or below the detection limit

2

of the SPR measurement. When exposed to undiluted FBS, the PEO brushes suppressed the

3

fouling to values below 10 ng·cm-2 (adsorbed amounts of less than 4% from the serum

4

deposits on pristine PDA films). The non-fouling characteristic is a result of the observed

5

effective screening of the charged, moderately hydrophilic and highly fouling PDA anchor

6

layer by polymer chains in the PEO(20000) brush, as well as the conformational freedom of

7

the swollen PEO chains as evidenced by the analysis of the streaming current data.

8 9

Table 3. Adsorbed protein mass on bare PDA and PEO-PDA films from single protein

10

solutions (HSA (5 mg·mL-1), Fbg (1 mg·mL-1) and Lys (1 mg·mL-1)) and undiluted FBS

11

measured by SPR. Layers

HSA

Fbg

Lys

FBS

[ng·cm-²] [ng·cm-²] [ng·cm-²] [ng·cm-²]

12 13

a

Bare gold

90 ± 10 270 ± 50 110 ± 20 300 ± 20

PDA

120 ± 30 290 ± 60 110 ± 30 270 ± 40

CH3O-PEO(20000)

-a

-

-

8±5

alkyne-PEO(20000)

1±1

4±4

4±3

9±7

Values below the detection limit of the SPR measurement i.e. < 0.7 ng·cm-2.

14

RGD functionalized PEO brushes. The PEO(20000) brushes mask the characteristics of the

15

PDA anchor layer and the substrate, thus providing the excellent non-fouling properties of the

16

whole PEO(20000)-PDA system. The alkyne derivate of the PEO(20000) brushes could be

17

successfully employed for the immobilization of RGD peptides bearing an azide group via

18

the CuAAC reaction. As determined by radio-assay measurements, the peptide surface

19

concentration (i.e. corresponding σpeptide and ) could be tuned from complete

20

saturation of the PEO surface with covalently bound peptides to values of the surface

21

parameters which might be relevant for cell adhesion studies (Table 1). In the same time the ACS Paragon Plus Environment

20

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Langmuir

1

radio imaging experiments proved macroscopically uniform immobilization of the RGD

2

sequences on the alkyne-PEO(20000)-PDA surfaces (SI, Figure S6). The presence of peptide

3

molecules on the alkyne-PEO(20,000) surfaces caused minute changes in the ellipsometric

4

thickness (on the level of the precision of SE measurement) and the hydrophilicity of the

5

layers in terms of water CA (Figure 4). The immobilization of 5.9–1.7×105 fmol·cm-2 RGD

6

peptides to the alkyne-PEO(20000)-PDA layers caused significant changes in the high

7

resolution core level C 1s, N 1s and O 1s XPS spectra (SI, Figures S7, S8 and S9) of these

8

surfaces. Beside the appearance of the amide C(=O)–NH contributions in the C 1s spectra at

9

288.2±0.3 eV, the presence of peptides caused gradual drop of the originally observed C–O–

10

C/C–C ratio for the alkyne-PEO(20000)-PDA from 5.5 to 2.4 at complete saturation of the

11

available alkyne groups. Alongside the grafting of RGD leads to gradual increase in the

12

surface concentration of tetrazole –NH–N=N– (398.4 eV) and amide NH–C(=O) (401.1 eV)

13

moieties (SI Table S1). The IRRAS measurements on the alkyne-PEO(20000)-PDA surfaces

14

bearing RGD could not unambiguously verify the immobilization of the peptides (SI, Figure

15

S10). The diminishing amount of surface immobilized moieties and the complex background

16

of the polymer stack made the quantitative measurements and qualitative assignment of the

17

bands in the differential spectra impossible. Therefore, to this end we only tentatively assign

18

the broad features in the 1700–1500 cm-1 region to the presence of amide I and amide II

19

vibrations of the surface immobilized peptides and the formed triazole rings42 on the course

20

of the CuAAC reaction. Nevertheless, the SE, CA, IRRAS and XPS measurements have

21

proven that the alkyne-PEO(20000)-PDA layers are stable during the course of the CuAAC

22

reaction. Streaming current measurements revealed a drop of the PZSC upon immobilization

23

of RGD to the brushes (Figure 5A). The PZSC values shifted from the initially observed

24

∼4.9 for the pristine alkyne-PEO(20000)-PDA to ∼4.1 for the layers bearing the highest

25

concentration (1.7×105 fmol·cm-2) of RGD peptides. Above the PZSC, the magnitude of


21

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Page 22 of 36

1

Istr/∆P increased with increasing surface concentration (i.e. surface density) of bound RGD

2

molecules (Figure 5A). At physiological pH (7.4), all RGD bearing layers showed a

3

monotonous increase in the magnitude of Istr/∆P compared to the initial values observed for

4

non-functionalized alkyne-PEO(20000)-PDA layers. The reasons for the increase of the

5

negative charge at the brush/electrolyte interface with increasing density of the RGD peptide

6

are not directly obvious from the streaming current measurements nor from the structure of

7

the RGD peptide. Since the PEO(20000) polymer chains provide a stable and effective

8

screening of the underneath PDA anchor layer and the supporting Si/SiO2 surfaces, we assign

9

the observed changes exclusively to the variations of the interfacial charge due to the

10

immobilization of the RGD peptides onto the brushes. The calculated pI of the peptide is

11

6.83.43 Accordingly the PZSC should be shifted to higher pH values with increasing RGD

12

surface concentration which is opposite to the experimental observation. To unravel the

13

reasons for the PZSC shift, we additionally measured the electrophoretic mobility of the

14

peptide in 1 mM KCl solution (SI, Figure S11). The point of zero electrophoretic mobility

15

(PZEM) was detected at pH ∼ 4.9. Because of the similarity of this value to the PZSC of the

16

PEO brushes, the shift of the PZSC towards pH ∼4.1 for the highest RGD surface

17

concentration cannot be explained without considering conformational changes of the peptide

18

upon attachment to the polymer chains in the brushes. The latter could be related to a

19

different pattern of charge compensation within the immobilized peptide and/or a different

20

accessibility to the counter charges in the streaming current experiment and thus to the

21

observed shift of the PZSC. Additionally, the unsymmetrical adsorption of water ions (OH-

22

>> H3O+)36, 44 could superpose the charging of the interface in presence of the RGD peptide.

23

The phenomenon is observed at uncharged, weakly charged and zwitterionic surfaces and

24

characterized by a negative excess charge at neutral and alkaline pH as well as electrical

25

neutrality of the interface around pH 4.44 As the streaming current measured for the highest


22

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Langmuir

1

RGD density shows a similar characteristic, the unsymmetrical adsorption of water ions

2

could increasingly contribute to the interfacial charge with increasing peptide surface

3

concentration.

4

In order to reproduce the streaming current data for the fully RGD functionalized brushes

5

(1.7x105 fmol⋅cm-2), we first considered the structural peptide charge resulting from the

6

ionization of functional groups in the amino acid sequence of the peptide (see Figure 5B).

7

The structural peptide charge was calculated using MarvinSketch 5.10.3 and assigned to the

8

PEO chain ends taking into account the chain end distribution shown in Figure 2B.

9

Furthermore, we assumed that the hydrodynamic penetration length was not altered after the

10

peptide immobilization (i.e., 1/λo = 18.3 nm). Using the structural peptide charge, the

11

simulations considerably overestimate the measured streaming current in the acidic and

12

alkaline pH region as well as the position of the PZSC (Figure 5A). According to the

13

theory45, a reduction of the hydrodynamic penetration length due to the peptide

14

immobilization would reduce the deviation between the predicted and measured data, but the

15

overall dependence of Istr/∆P vs. pH would be similar to the curve shown in the inset of

16

Figure 5A i.e., a reproduction of the measured PZSC would be not possible by the variation

17

of 1/λo.

18

Next, we determined the pH dependence of the electrokinetic active peptide charge that

19

best reproduces the measured streaming current for the completely RGD functionalized brush

20

(1.7×105 fmol⋅cm-2). The charge that allowed a complete reproduction of the experimental

21

data was found to be significantly lower as compared to the structural peptide charge and

22

shows a similar pH dependence as the measured streaming current (Figure 5B). Furthermore,

23

the shape of the curve with the PZSC around pH 4 supports the assumption that

24

unsymmetrical adsorption of water ions44 contributes to the charging of the RGD

25

functionalized brushes. To reproduce the streaming current vs. pH data measured for the


23

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Page 24 of 36

1

brushes with the lower RGD densities, we reduced the electrokinetic active charge according

2

to the RGD densities given in Table 1. This strategy did not allow a consistent fit of the

3

streaming current vs. pH data shown in Figure 5A for surface concentrations below 1.7×105

4

fmol⋅cm-2. For all RGD concentrations below this value the curves were found to be similar

5

to the curves measured/calculated for the PDA anchoring layer. These results show that the

6

interfacial charge formation does not directly correlate with RGD functionalization of the

7

PEO brush. Instead the results suggest that the interfacial charging results from pH-dependent

8

contributions of the PDA anchor layer, the ionization of functional groups in the amino acid

9

sequence of the RGD and unsymmetrical water ion adsorption. According to the

10

experimental data, the latter might already dominate the interfacial charging at the brush with

11

the lowest RGD density.

12

The adsorption of globular proteins from solution onto a surface is related to the proteins

13

native structure and the physicochemical characteristic of the surface. The presence of

14

peptides at the distal-end of the performed non-fouling layers might have a detrimental effect,

15

tuning the initially non-fouling layer to highly fouling. Current biomimetic surface

16

engineering has been focused on deciphering the importance of peptide ligand specificity,

17

density, topographical and spatial arrangement on cell behavior.46-50 However, the changes in

18

the fouling behavior of polymer brushes by the incorporation of bioactive molecules have

19

been overlooked or circumvented by following the cell/artificial ligand interactions in a

20

serum free media, providing rather unnatural initial physiological environment. In order to

21

probe the influence of exerted physico-chemical changes by the immobilization of the RGD

22

motifs on the non-fouling properties of the layers and thus probe the sole behavior of these

23

biomimetic layers in biological media used as cell cultivation supplements, we have

24

performed protein adsorption studies as in the case of the neat PEO(20000)-PDA films.

25

Generally, the presence of the integrin selective peptide motifs at the PEO distal-end affected


24

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Langmuir

1

the initially observed resistance to fouling of the neat PEO(20000)-PDA films to a minor

2

extent (Figure 6). This is in line with the electrokinetic measurements which proved that the

3

thick swollen PEO brush screens the negative charge of the PDA anchor layer. Furthermore,

4

the data suggest the phenomenon of presumed unsymmetrical water ion adsorption

5

determines the interfacial charging at peptide concentrations ≥600 fmol·cm-2. The fouling

6

from the structurally unstable “soft" and negatively charged proteins, i.e. HSA and Fbg

7

showed no particular dependence on the concentration of immobilized peptides. The

8

adsorbed mass of HSA and Fbg reached values of up to 20 ng·cm-2 corresponding to less than

9

20% and 7% of the respective monolayers formed on bare gold (Figure 6A and B). It can be

10

assumed that electrostatic interactions with positively charged patches present on the protein

11

surfaces bearing overall negative charge together with van-der-Waals forces and hydrogen

12

bonds guide the adsorption of HSA and Fbg on the RGD-PEO(20000)-PDA surfaces.

13

A significant increase in mass of adsorbed proteins with increasing concentration of bound

14

RGD peptides was observed when Lys was used as an analyte (Figure 6C). Such an

15

observation is in line with the expected electrostatic attraction between the positively charged

16

and internally coherent molecules of Lys and the negatively charged moieties in the RGD

17

sequences on the PEO(20000)-PDA surfaces. At complete saturation of the alkyne-

18

PEO(20000)-PDA surfaces with RGD peptides (1.7×105 fmol·cm-2), the mass of adsorbed

19

Lys reached a value of 50 ± 30 ng·cm-2. Notably, the herein presented PEO(20000)-PDA

20

layers bearing RGD adhesion motifs in the range 5.9–1.7×105 fmol·cm-2 managed to reduce

21

the fouling from FBS to less than 30 ng·cm-2 (corresponding to less than 10% of the deposits

22

formed on bare gold) (Figure 6D) For comparison, the FBS deposits on pristine alkyne-

23

PEO(20000)-PDA surfaces were 9 ± 7 ng·cm-2. Layers exhibiting such a low-fouling are

24

reasonably expected to sufficiently elicit the specific artificial surface-integrin receptors

25

interactions even in the presence of cultivation media, i.e. FBS. The possibility to design and


25

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Page 26 of 36

1

selectively bind the biomimetic peptide ligand on the distal ends of the anchored polymer

2

chains makes the PEO-PDA surface modification a robust platform for the surface

3

engineering of different biomedical and tissue engineering devices.

4 5


26

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Langmuir

1 2

Figure 4. Dependence of water contact angles of RGD bearing PEO(20000)-PDA layers on

3

surface concentration of RGD peptides. The measurements were performed on layers

4

performed on gold SPR chips (circles) and Si/SiO2 substrates. The range of water contact

5

angles of alkyne-PEO(20000)-PDA layers is given by the horizontal lines (average value ±

6

standard deviation) for comparison (line).

7


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Page 28 of 36

1

2 3

Figure 5. Streaming current over applied pressure, Istr/∆P, as a function as a function of the

4

pH in 1 mM KCl solution for pristine and RGD functionalized alkyne-PEO(20000)-PDA

5

brushes (A). The symbols pertain to experimental data and the solid lines represent

6

simulation curves. The grey and black curves were obtained for fully RGD-functionalized

7

brushes (1.7×105 fmol·cm-2) bearing either the structural peptide charge (grey) or the

8

electrokinetic active peptide charge (black) shown in frame B. The blue line represents the

9

simulation data for the pristine PEO brush (compare to Figure 3). All other simulation curves

10

were obtained for the RGD functionalized brushes assuming a reduction of the

11

electrokinetically active RGD charge (shown in frame B) by a factor given by the ratio of the

12

respective RGD concentration to the maximum RDG concentration (1.7×105 fmol·cm-2). The

13

intersection of the curves shown in frame A with the horizontal line at Istr/∆P = 0 corresponds

14

to the PZSC.

15


28

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Langmuir

1 2 3

Figure 6. Adsorbed protein mass from HSA (A), Fbg (B), Lys (C), and FBS (D) versus the

4

surface concentration of immobilized RGD peptides. The adsorption to alkyne-PEO(20000)-

5

PDA layers is reported for comparison (Line). The bare gold surfaces used in this study

6

revealed accumulation of 90 ± 10, 270 ± 50, 120 ± 30, and 290 ± 60 ng·cm-2 of adsorbed

7

HSA, Fbg, Lys, and FBS, respectively. The fouling on PDA surfaces was on the same level

8

of adsorbed protein mass as in the case of bare gold (Table 3). The detection limit of the

9

measurements was 0.7 ng·cm-2. All measurements have been performed at pH = 7.4.

10


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Page 30 of 36

1

CONCLUSIONS

2

PEO brushes that suppress protein adsorption and provide at the same time various options

3

for biomimetic surface engineering were prepared by dense end-tethering of α-aminoethyl

4

capped CH3O-PEO(20000) and alkyne-PEO(20000) chains to PDA-modified substrates.

5

Spectroscopic ellipsometry, IRRAS, and XPS verified the formation of covalently anchored

6

and densely packed PEO layers (σ = 0.5 ± 0.1 for CH3O-PEO(20000) and 0.7 ± 0.1 for

7

alkyne-PEO(20000) [chains·nm-2]) with a pronounced overlapping of the tethered chains

8

leading to a brush structure, i.e.

9

the basis of combined SCF and soft surface electrokinetic theories provided detailed insights

10

into the polymer segment density distribution in the brushes, the interfacial charging, and the

11

charge screening by the immobilized PEO chains, the latter leading to the pronounced non-

12

fouling character of the coatings.

!"

= 0.1. Evaluation of streaming current measurements on

13

The coupling of bio-adhesive RGD peptide ligands to alkyne-PEO(20000) brushes by

14

CuAAC led to minute changes in the SE, CA and IRRAS data, but could be unambiguously

15

verified by XPS. To our best knowledge, we additionally utilized for the first time

16

electrokinetic measurements and theoretical predictions for monitoring and unraveling the

17

charging effects introduced to a polymer brush system by the immobilization of peptides in a

18

large surface density region. Streaming current measurements performed on these

19

macroscopically uniform surfaces showed a continuous drop in the PZSC from the initially

20

observed 4.9 for the neat films to ∼4.1 for the layers bearing and the highest surface

21

concentration of 1.7×105 fmol·cm-2 of RGD peptides. Above the PZSC, the magnitude of

22

Istr/∆P increased with increasing surface concentration (i.e. surface density) of bound RGD

23

molecules, leading to higher streaming currents than the ones observed for the pristine

24

alkyne-PEO(20000)-PDA surfaces. The theoretical predictions suggest that the observed

25

trends in the interfacial charging might result from the unsymmetrical adsorption of water


30

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Langmuir

1

ions (OH- >> H3O+), as well as the pH-dependent ionization of the PDA anchor layer and

2

structural peptide charge.

3

The presence of the integrin selective peptide motifs affect the initially observed resistance

4

of the neat alkyne-PEO(20000)-PDA surfaces towards fouling from HSA and Fbg to a minor

5

extent with no particular dependence on the concentration of immobilized RGD peptides. In

6

line with the expected electrostatic attraction between the positively charged and structurally

7

stable molecules of Lys and the negatively charged RGD-PEO(20000)-PDA surfaces, the

8

mass of adsorbed Lys increased with increasing concentration of surface bound RGD

9

peptides. Strikingly, the layers decorated with RGD adhesion motifs reduced the fouling in

10

FBS containing media to values comparable with the ones measured for pristine alkyne-

11

PEO(20000)-PDA surfaces. In sum, peptide decoration was found to cause only minor

12

changes of the excellent protein repellency of PEO(20000)-PDA coatings which

13

recommends this system for further studies using libraries of alternative peptide motifs.

14 15

ASSOCIATED CONTENT

16

Supporting Information.

17

Experimental details, segment density profiles, detailed IRRAS and XPS analysis PEO-PDA

18

layers covalent structure high resolution core level C 1s, O 1s, N 1s spectra of PDA anchor,

19

CH3O-PEO(20000), alkyne-PEO(20000) and RGD peptides bound to alkyne-PEO(20000)-

20

PDA layers; Radiography scans, IRRAS spectra and Electrophoretic mobility of free RGD

21

peptides are available as a single pdf file.

22

This material is available free of charge via the Internet at http://pubs.acs.org

23


31

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1

AUTHOR INFORMATION

2

Corresponding Author

Page 32 of 36

3

* Ognen Pop-Georgievski, Institute of Macromolecular Chemistry, Academy of Sciences of

4

the Czech Republic, Heyrovsky sq. 2, Prague 6, 162 06, Czech Republic, phone number:

5

+420-296809225, fax number: +420-296809410, e-mail: [email protected]

6

* Ralf Zimmermann, Leibniz Institute of Polymer Research Dresden, Max Bergmann Center

7

of Biomaterials Dresden, Hohe Str. 6, 01069 Dresden, Germany, phone number: +49 (0)

8

3514658258, fax number: +49 (0) 3514658533, e-mail: [email protected].

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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENT

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The support from the Grant Agency of the Czech Republic (GACR) under Contract No. 15-

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09368Y and 16-02702S, from the European Regional Development Fund OPPK

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(CZ.2.16/3.1.00/21545) is gratefully acknowledged. J.K. acknowledges the support from the

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Ministry of Health of the Czech Republic (Grant # 16-30544A).

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Impact of Bioactive Peptide Motifs on Molecular Structure, Charging

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