Layer-by-Layer Assembly of Heparin and Peptide-Polyethylene Glycol

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Layer-by-Layer assembly of heparin and peptide-polyethylene glycol conjugates to form hybrid nanothin films of biomatrices Alvin Kuriakose Thomas, Robert Wieduwild, Ralf Zimmermann, Weilin Lin, Jens Friedrichs, Marc Bickle, Karim Fahmy, Carsten Werner, and Yixin Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02014 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018

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ACS Applied Materials & Interfaces

Layer-by-Layer assembly of heparin and peptidepolyethylene glycol conjugates to form hybrid nanothin films of biomatrices Alvin Kuriakose Thomas 1, Robert Wieduwild 1†, Ralf Zimmermann 2, Weilin Lin1, Jens Friedrichs 2, Marc Bickle 3,Karim Fahmy 4, Carsten Werner 2, Yixin Zhang* 1 1

B CUBE Center for Molecular Bioengineering, Technische Universität Dresden, Dresden,

Germany 2

Department of Biofunctional Polymer Materials – Max Bergman Center of Biomaterials,

Leibniz Institute of Polymer Research, Dresden, Germany 3

High-Throughput Technology Development Studio, Max Planck Institute of Molecular Cell

Biology and Genetics, Dresden, Germany 4

Helmholtz-Zentrum Dresden-Rossendorf, Institute of Resource Ecology, Dresden, Germany

and Biotechnology center (BIOTEC), Technische Universität Dresden, Dresden, Germany

KEYWORDS: extracellular matrix, layer-by-layer, nano thin, heparin, biomimetic.

ACS Paragon Plus Environment

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ABSTRACT: We investigated the utility of a heparin/peptide-polyethylene glycol conjugate system to build Layer-by-Layer (LbL) structures, to assemble tailored multilayer-biomatrices for cell culture. The LbL assembly balances the advantages of polyelectrolyte systems and proteinbased systems. Human umbilical vein endothelial cells showed distinct responses to: the film thickness and structure; the presence, density and spatial arrangement of a cell adhesion ligand within the nanothin film; and the pretreatment of the film with morphogens. The LbL technique presents a versatile tool for modifying cell culture substrates with defined and diverse biochemical and structural features, for investigating cell-material interactions.

Biomimetic materials play a crucial role in current efforts towards studying cell biology, and enabling tissue engineering and regenerative medicine. Cell culture substrates have evolved from simple polystyrene surfaces to proteins coatings, extracellular matrix (ECM) extracts or complex 2D and 3D systems, in order to recapitulate various aspects of the organization and multicellular complexity of tissues.1–3 While many 2D coatings are useful for investigating individual biochemical cues, 3D biomatrix based cell culture presents a promising avenue to model the pathophysiological environments of tissues.

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However, the shift from simple 2D tissue culture

plastic surface to 3D structure with tailored functions has raised challenges and complications.5 For example, aggregation, instability, batch-to-batch differences, immunogenicity, and contamination could be intrinsic problems for some protein-based materials, especially for the clinical applications. The promiscuous interactions between proteins and surfaces can also cause complications for biomaterial engineering, for example, to Layer-by-Layer (LbL) assemble biomatrix films as discussed in this work. LbL growth of a biomatrix film would allow us to investigate cell material interaction, through a gradual transition from 2D surface coating to form a more complex matrix.


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Poly-lysine/poly-ornithine or different oligosaccharides based polyelectrolyte multilayers (PEMs) have been tested for their utility as simplified systems to interrogate cell material interactions.6–10 To incorporate proteins into LbL system will provide the matrix with biological functions such as enhanced cell adhesion, but on the other hand, cause the loss of linear growth in the LbL process. Proteins, immobilized on surface or diffusing in solution, have multiple differently charged and hydrophobic areas and thus can cause many non-specific interactions with other molecules or surfaces. Protein absorption and coating on a surface are mainly mediated by such promiscuous interactions. The promiscuous interaction will complicate an LbL system, for example, causing unwanted exponential growth through the cycles. LbL growth restricted to a patterned surface is thus very difficult. For example, LbL build up employing one of the most specific and potent protein ligand interactions: the streptavidin biotin pair did not lead to stable, controlled and sustainable LbL development.11 Herein, we describe an LbL system, which can be built up in a controlled manner like the classical polyelectrolyte systems as well as incorporate biochemical functionalities. To use peptide and oligosaccharide as an interacting pair could have following major advantages: i) to tune the peptide oligosaccharide interaction to achieve optimal stepwise LbL growth; ii) to introduce peptide based functionalities into the film; iii) to realize structural simplicity in order to diminish the aggregation and unspecific absorption associated with the large proteins. Short ECM-mimicking peptides are chemically more defined, less immunogenic, and can mimic many desired functions of large proteins e.g., fibronectin, laminin, collagen; 12–14 iv) Oligosaccharides are essential components in ECM and some of them like heparin and dextran sulfate have also been approved for medical uses by the regulatory agencies. We envisioned that an LbL system, based on a non-covalent interaction between peptide and


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oligosaccharide, could lead to an attractive technology for bottom up assembly of tailored biomatrices, from simple 2D surface coating to form more complex structures based on previous studies. 8,15,16

Figure 1. Principle and characterization of the peptide-starPEG/heparin LbL system. a) Principle and components used for the LbL assembly. b) QCM measurements of a composite film containing 30 layers. The arrows indicate the time point of injection of heparin or the respective peptide-starPEG conjugate into the fluidic system. c) Dissipation measurements for 12 layers of the peptide-starPEG/heparin systems: KA7-starPEG/heparin (KA7), ATIIIstarPEG/heparin (ATIII) and RA7-starPEG/heparin (RA7). d) Electrokinetic measurements of streaming current vs. pressure gradient measured for each deposition step during the built-up of heparin and RA7-starPEG film.


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If the LbL assembly is mediated by specific interaction between simple chemical structures, we could have the opportunity to diminish the effect caused by non-specific interaction. We have previously developed a physical hydrogel system assembled by the interaction between sulfated oligosaccharides and peptide-polymer conjugates.

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The minimal heparin binding motif (BA)n

(or BAn) where B is a basic residue lysine (K) or arginine (R), A is alanine, and n is the number of repeats, which enables us to tune the stiffness and gelation rate of the non-covalent networks. The specificity between BAn and heparin is also associated with a conformational change of the peptide from random coil to ordered secondary structures upon binding to heparin. For developing an LbL system, we have chosen KA7 and RA7 (Figure 1), as well as a heparin binding peptide with a more complex sequence (ATIII peptide from anti-thrombin). While the gelation of KA7-starPEG/heparin is slower than RA7-starPEG/heparin, ATIII-starPEG and heparin form amorphous particles. 17 We investigated whether the peptide-starPEG conjugate and heparin can be used for the LbL assembly of nano thin films, and the structure can prevent the promiscuous interaction often associated with protein based materials (Figure 1a). To grow the biomatrix in an LbL manner and characterize the development in situ, we covalently immobilized heparin (as layer 1) onto the surface of QCM (quartz crystal microbalance) chip, followed by adding the peptide-starPEG and heparin alternately (SI). As shown in Figure 1b, frequency shifts have demonstrated the LbL growth of KA7-starPEG, or RA7-starPEG, or ATIII-starPEG with heparin, whereas repetitive injections of single component (either peptide-starPEG or heparin) did not lead to growth (SI). The dissipation values remained relatively stable when ATIII-starPEG or KA7-starPEG was used, whereas the LbL growth using RA7-starPEG has caused a remarkable increase in dissipation, indicating softer matrix films (Figure 1c, SI). Biomaterial stiffness could be


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carefully tuned by controlling the number of the layers deposited while to coupling it the mechanical properties of the substrate below.

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The matrix can grow in a hybrid fashion, with

layers 1 to 10 of heparin/RA7-starPEG, layers 11 to 20 of heparin/ATIII-starPEG, and layer 21 to 30 of heparin/KA7-starPEG developed consecutively (Figure 1b, SI). The LbL assembly of hybrid films could also be confirmed by using different fluorescently labelled peptide-starPEGs (SI). The interfacial charge of the films through LbL growth was followed by using streaming current measurements at a neutral pH with micro slit electrokinetic cell (Figure 1d, SI), to further demonstrate that the growth of each layer changes the matrix surface and prepares for the following step when heparin and peptide-starPEG were added alternately. As expected, the immobilization of the negatively charged heparin to the positively charged aminosilane modified substrate caused a sign reversal of the streaming current to negative values (Figure 1d), thus confirming the successful immobilization of heparin to the aminosilane layer. In the subsequent deposition steps the streaming current oscillated between positive and negative values when peptide-starPEG and heparin were added alternately (Figure 1d, SI). We then followed the LbL growth of matrix film using atomic force microscope (AFM) (Figure 2a). The films were grown by alternatively depositing the heparin and peptide-starPEG. We measured the film thickness after the first layer of covalently bound heparin (Layer 1H), Layer 2P (second layer of peptide starPEG) and subsequently at Layer 6P, Layer 10P, Layer 14P, and Layer 18P with AFM after scratching the film (SI). In good agreement with the QCM measurements, the buildup of RA7-starPEG/heparin system was exponential. Exponential growth of films have often been observed in other LbL systems such as collagen/heparin or poly (L-glutamic acid) /poly (L-lysine), indicating a less compact structure that permits the diffusion


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of one or both polymers.

8,19,20

The increase of dissipation in QCM measurement also indicates

that LbL buildup of RA7-starPEG/heparin film resulted in softer matrix. In contrast, when KA7starPEG was used, a linear growth of film thickness has been observed, while ATIII-starPEG has shown a behavior intermediate to RA7-starPEG and KA7-starPEG. The stepwise growth of films were associated with the increase of surface roughness and changes in surface topology. Growing KA7-starPEG/heparin and ATIII-starPEG/heparin films led to the gradual increase of fibrous structures, while the surface roughness also increased gradually (Figure 2b and Table S1). Upon increasing layer number, the topology of RA7-starPEG/heparin film changed from fibers to larger domains, and the overall surface roughness also increased dramatically (Figure 2c). Different from the proteins used for surface coating, the peptide-starPEG conjugates and heparin showed minor absorption on plastic or glass surfaces, while their absorption on PEGylated glass surface is very low. This feature allowed us to grow the biomatrix film on a patterned surface. We patterned a PEGylated glass surface with fluorescently labeled heparin (SI). KA7-starPEG and heparin were added alternately and AFM was used to follow the film build up. A clear boundary has been observed through the LbL growing process, while only minor amounts of material deposition on the PEGylated surface has been detected starting at the ninth layer.


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Figure 2. AFM characterization of the LbL films. a) Plot for film thickness and surface roughness (Ra) vs. layer number obtained from AFM measurements in PBS in a fluidic cell. The first layer is heparin. b) Topography of a 15 µm2 region the LbL films for different peptidestarPEG/heparin in. c) Patterned development of LbL film. The AFM image of the LbL of the RA7 starPEG/heparin system at the boundary of the pattern, developed on a PEG passivated surface at layer 1 and layer 9. Scale bar is 5 µm.


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ATIII-starPEG and RA7-starPEG could also be used to grow films on patterned surface, while both conjugates have slightly higher tendency to form depositions on PEGylated areas (SI). ATIII-peptide has a more complex sequence and the guanidinium group of arginine can cause larger number of electrostatic interactions compared to lysine.

17,21,22

Therefore, KA7-

starPEG/heparin is the optimal combination for controlled LbL growth. The specific interaction between the simple chemical motifs minimizes the promiscuous interaction and non-specific absorption associated with many protein based materials. We have shown that hybrid structures and various topologies could be created through LbL growth of the peptide starPEG/heparin system. It would allow us to investigate cell material interaction, through a gradual transition from 2D surface coating to more complex matrix. Many types of cells cannot adhere to the KA7-starPEG/heparin and RA7-starPEG/heparin hydrogels. 17,23

Arg-Gly-Asp-Ser-Pro (RGDSP) is an effective and well-established, cell adhesion

promoting peptide ligand. 24 We synthesized RA7-RGDSP-starPEG and placed the cell adhesive building block as a layer in the nano thin film. A heparin modified surface (Layer 1H) in a fluidic device was treated with either RA7-starPEG (L2P) or RA7-RGDSP-starPEG (L2 RGDSP). Human Umbilical Vein endothelial Cells (HUVECs) did not adhere to the Layer 2P surface, but as expected, cell adhesion was dramatically improved on Layer 2 RGDSP (SI). RA7-RGDSP-starPEG was mixed with RA7-starPEG in different ratios and placed at Layer 2. A dose dependent response of HUVEC to the presented adhesion ligand RGDSP at the second layer, was observed (Figure 3a). Moreover, we could not only tune the density of cell adhesive peptide in the nano thin hybrid biomatrix, but also control its spatial location. We grew four, eight, or 14 layers using RA7-starPEG/heparin on the Layer 2 RGDSP. Additional layers without the cell adhesive sequence shelled the effect of Layer 2 RGDSP and reduced the cell attachment


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gradually (Figure 3b). Then we tested the effect of RA7-RGDSP-starPEG above different numbers of RA7-starPEG/heparin layers. Interestingly, the cell adhesion decreased when the matrix became thicker (Figure 3c). Image analysis revealed that the loss of adhesion was associated with reduced area per cell and increased roundedness (Figure 3d). The observation is very similar to the behavior of HUVEC on LbL assembled multilayer films built by DNA and protamine sulfate. An increase in bilayer number leads to a decrease in stiffness as well as the adhesion of HUVEC.25 The influence of LbL build up on cell morphology was less pronounced when KA7-starPEG was used, while ATIII-starPEG showed an effect similar to that of RA7starPEG. HUVECs attach to stiff surfaces better than to soft surfaces

9,15,26,27

. The LbL buildup

of RA7-starPEG/heparin resulted in a thick and soft matrix film, as shown by the AFM and QCM dissipation measurements (Figure 1c, Figure2a and Figure2b). Therefore, the different responses of cells could be attributed to the gradual change in the mechanical properties of nano thin films. Heparin based hydrogels has recently attracted much interest because the resulting biomaterials could function as a morphogen storage and releasing system

15,23,26,28,29

. It was therefore was

important to test the effect of morphogen loading on cell adhesion. Interestingly, whereas FGF pretreatment of RA7-starPEG/heparin film had no effect, VEGF pretreatment improved cell adhesion (Figure 3e). Moreover, VEGF or FGF pretreatment and the display of cell adhesive sequence (RA7-RGDSP-starPEG/heparin film) have shown synergetic effects on cell morphology (SI). Similar to the bulky heparin containing hydrogels, we can combine the adhesion stimulus with the morphogen stimulus in the LbL nano thin film.


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Figure 3. Human Umbilical Vein Endothelial Cells (HUVECs) on LbL films. a) Effect of adhesion ligand; RGDSP, density was tested by plotting the dose dependent cell adhesion response of HUVECs by mixing RA7-starPEG and RA7-RGDSP-starPEG in different ratios at Layer 2. b) Barrier Effect; The first covalently attached layer of RA7-RGDSP-starPEG was followed by growing RA7-starPEG/heparin LbL films to create a barrier between the adhesion ligand RGDSP and the cell . Scale bar: 200 µm. c + d) Position of adhesion ligand; Effect of the placement of the cell adhesive ligand RGDSP, at the top of multiple layers for KA7-starPEG/ heparin LbL systems. Shown here is the response of cell at the 6th and 10th layers. Scale bar: 200 µm. e) Morphogens Storage and Release of RA7-starPEG/heparin film. The system was preincubated with the growth factors (from left to right: none, FGF and VEGF) followed by rinsing with PBS buffer. Cells were cultured in minimal cell culture medium. Scale bar: 200 µm.


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ECM can provide different mechanical and biochemical supports to cells, e.g., bulky matrix filling interstitial space, or nano thin layer such as basement membrane. Development of ECM mimicking biomaterials aims to recapitulate the native environment of different tissues. Various cues of biomaterials affecting HUVEC adhesion, including the presence of integrin binding ligand, growth factors storage and structural property have been previously illustrated on bulky hydrogels. Through using the LbL modular system, we have shown that similar rules can also be applied to nano thin films. The technology can provide a versatile platform for the screening and design of tailored materials for culturing cells, in addition to various 2D coating and bulky 3D hydrogel materials.

Figure 4. A versatile LbL technology to buildup nanothin films of biomatrices


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In summary, we describe an LbL buildup of nano thin film system, which can be engineered as tunable hybrid structures, grown on patterned surface, and function as morphogen storage and release system (Figure 4). It represents a versatile tool for studying cell material interaction, starting from a simple 2D surface coating, shifting gradually to more complex forms. By using the LbL bottom up method, many complex features of the matrix network, including thickness, morphology and ligand densities could be tuned. The simplicities in both chemical composition and engineering procedure as well as the great variety of tunable properties could lead to deeper understanding of not only of the interaction of cells with the ECM in general, but also the various mechanisms of how different cells adapt to their particular micro environments. In the future, other factors such as mechanical stress in fluidic microenvironments, different cell adhesion ligands and glycosaminoglycans, as well as various types of cells will be investigated using this system.30


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publication website at doi: xx.xxx/acsami.xxxxxxx The synthesis procedure and characterization data of synthesized components required for the nanothin films. The experimental setup and assays details for the characterization of the nanothin films. The chemicals, materials and the equipment used for the experiments. (PDF) AUTHOR INFORMATION Corresponding Author Yixin Zhang, B CUBE Center for Molecular Bioengineering, Technische Universität Dresden, Arnoldstraße 18, 01307 Dresden, Germany E-mail address: [email protected] Present Addresses † Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by the Leibniz Association; SAW-2011-IPF-2 and BMBF; BioLithoMorphie 03Z2E512.


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ACKNOWLEDGMENT We would like to thank Ulrike Hofmann – B CUBE Center for Molecular Bioengineering, Suzanne Bartsch and Juliane Drichel - Max Bergman Center of Biomaterials for technical support. We thank also thank Silke White and Isabel Raabe from the Imaging Platform of the Deutsches Zentrum Für Neurodegenerative Erkrankungen. We would like to thank Dr. Tilo Lübken, NMR-Service of the Organic Chemistry department of the Technische Universität Dresden for the NMR measurements. Stephanie Möllmert, Shada Abuhattum and CMCB Technology Platform are thanked for help with AFM measurements. The authors declare no conflict of interest or competing interests. ABBREVIATIONS Layer-by-Layer, LbL; Extracellular Matrix, ECM; Polyelectrolyte Multilayers, PEMs; Atomic Force Microscope, AFM; Arg-Gly-Asp-Ser-Pro, RGDSP; Human Umbilical Vein Endothelial Cells, HUVECs; Supporting Information, SI.


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Glycosaminoglycan-Based

Hydrogels

Capture

Inflammatory Chemokines and Rescue Defective Wound Healing in Mice. Sci. Transl. Med. 2017, 9 (386), eaai9044. (30)

Minami, K.; Mori, T.; Nakanishi, W.; Shigi, N.; Nakanishi, J.; Hill, J. P.; Komiyama, M.; Ariga, K. Suppression of Myogenic Differentiation of Mammalian Cells Caused by Fluidity of a Liquid–Liquid Interface. ACS Appl. Mater. Interfaces 2017, 9 (36), 30553– 30560.


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Layer-by-Layer Assembly of Heparin and Peptide-Polyethylene Glycol

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