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The journey of supramolecular polymers to biomaterials : from

fundamental studies to applications

Citation for published version (APA):

Hendrikse, S. I. S. (2018). The journey of supramolecular polymers to biomaterials : from fundamental studies to applications. Technische Universiteit Eindhoven.

Document status and date: Published: 18/10/2018 Document Version:

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The Journey of Supramolecular Polymers to

Biomaterials: From Fundamental Studies to

Applications

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Technische Universiteit

Eindhoven, op gezag van de rector magnificus prof.dr.ir. F.P.T. Baaijens,

voor een commissie aangewezen door het College voor Promoties, in het

openbaar te verdedigen op donderdag 18 oktober 2018 om 16:00 uur

door

Simone Iris Sandra Hendrikse

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voorzitter: prof.dr. P.A.J Hilbers 1e promotor: prof.dr. E.W. Meijer 2e promotor: prof.dr.dr. P.Y.W. Dankers leden: prof.dr. M.P. Lütolf (EPFL)

prof.dr. R.J.M. Nolte (RUN) prof.dr.ir. L. Brunsveld adviseur(s): dr.ir. M.M.C. Bastings (EPFL)

dr. M.B. Baker (MU)

Het onderzoek of ontwerp dat in dit proefschrift wordt beschreven is uitgevoerd in overeenstemming met de TU/e Gedragscode Wetenschapsbeoefening.

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For the high and lows And moments between Mountains and valleys, And rivers and streams For where you are now And where you will go For “I’ve always known”

And “I told you so” For “nothing is happening”

And “all is wrong” It is here in this journey You will learn to be strong You will get where you are going

Landing where you belong ̴ Morgan Harper Nichols ̴

The only failure is not to explore at all ̴ Sir Ernest Shackleton in Shackleton ̴

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Cover design: Simone Hendrikse Printed by: Gildeprint, Enschede

A catalogue record is available from the Eindhoven University of Technology Library ISBN: 978-90-386-4594-0

This work was funded by the NWO/DPI program NEWPOL (Project No. 731.015.503), partially financed with Topconsortia for Knowledge Innovation allowance provided by the Dutch Ministry of Economic Affairs.

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Chapter 1 From ECM inspiration to functional biomaterials ... 1

Chapter 2 Tuning the dynamic nature of supramolecular polymers ... 19

Chapter 3 Probing the adhesion of supramolecular polymers to cells ... 31

Chapter 4 A supramolecular platform stabilizing growth factors ... 41

Chapter 5 Glycocalyx mimicking co-assembled BTA-based polymers ... 55

Chapter 6 Installing antimicrobial activity in a multivalent display ... 71

Chapter 7 Designing a niche for intestinal organoid expansion ... 81

Chapter 8 Evaluating supramolecular hydrogels for liver organoid expansion ... 95

Chapter 9 Epilogue ... 103

Summary ... 113

Curriculum Vitae ... 117

List of publications ... 119

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F R O M E C M I N S P I R A T I O N T O

F U N C T I O N A L B I O MA T E R I A LS

The complexity of the extracellular matrix (ECM) in terms of structure, components, interacting cues, and function, inspired scientists to mimic these properties in biomaterials. Depending on the area of application, elastomeric biomaterials can be used as load bearing scaffolds for in vivo transplantation, whereas hydrogels are proposed to support in vitro cell culture. Although the chemical structures and systems designed and studied today are rather simple compared to the complexity of the ECM, first examples of these functional supramolecular biomaterials reaching the clinic have been reported. Dynamic fibrous supramolecular assemblies in hydrogels are in particular interesting due to their ability to support cell expansion in a responsive and adaptable fashion similar to the natural situation. Due to its modular properties, multi-component assemblies can be formed to assess minimal requirements for cell culture. In this chapter a short introduction is provided on biomaterials with a focus on functional fibrous supramolecular hydrogels for regenerative medicine applications.

Part of this chapter has been published in a review:

O.J.G.M. Goor,* S.I.S. Hendrikse,* P.Y.W. Dankers, E.W. Meijer. From supramolecular polymers to multi-component biomaterials. Chem. Soc. Rev., 2017, 46, 6621-6637.

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1.1 Introduction

The cellular environment is a source of inspiration for the design of artificial mimics for regenerative medicine applications since it orchestrates cell behavior in a dynamic yet spatiotemporal manner.1 The interwoven fibrous network of the extracellular matrix (ECM) is extremely challenging to mimic, due to its extreme complexity and cell-type dependent properties. So far major progress is being made in arriving at simplified yet functional materials guiding cell behavior similar to the in vivo ECM. Unfortunately, one material does not fit every requirement since every cell type is distinct, contains unique protein compositions and isoforms, and therefore requires cell type specific material properties. Another important property of the ECM is its structure. The ECM consists mainly of fibrous proteins, like collagen, laminin and elastin, and non-fibrous proteins like proteoglycans containing glycosaminoglycan (GAG) chains.2, 3 Collagens are self-assembled into either fibrils, associate with fibrils, or form networks, providing structural support, whereas laminins polymerize upon activation through binding certain integrins, important in cell adhesion. Also fibronectins are known to assemble into fibrils upon activation by integrins and align with intracellular actin stress fibers. In addition, elastin provides elasticity due to crosslinking and nidogen-1 connects laminin to collagen type IV. In contrast to fibrous ECM proteins, glycosaminoglycans are linear carbohydrate-based polymers, which are highly negatively charged due to hydroxylate, carboxylate and sulfate groups and are able to bind many water molecules and different proteins. This strong solvability and the high water content facilitate high resistance to compressive forces, allow diffusion of various bioactive compounds, and stabilize and present growth factors.

All ECM proteins can exist in different isoforms that have slightly different functions. However, the common property is their ability to bind other ECM proteins by non-covalent interactions. Therefore, the ECM is an interconnecting network that links various ECM proteins together and to the cell surface via specific receptors such as the integrin receptors. The ECM can in general be divided in various types that are tissue and/or organ specific. Furthermore, the ECM is different in structure and composition at the cell surface (pericellular matrix), in epithelial and endothelial tissues (basement membrane), and in connective tissues (interstitial ECM). The basement membrane is a thin, dense area at the cell-ECM interface that is highly enriched in collagen type IV, laminin, perlecan and nidogen.3 It is known as the specialized ECM region which also regulates the cell behavior. Integrin receptors present in the cell membrane connect the ECM with the intracellular cytoskeleton.4, 5 Since integrins have the remarkable property to signal bidirectionally, they are able to induce both intracellular and extracellular changes due to ECM or intracellular stimuli, respectively. In order to tightly bind to ECM components, multiple activated integrins must cluster together to form focal adhesions.6 The subsequent signal transduction conducted by integrins also occurs in response of physical forces, known as mechanotransduction.7 Mechanical forces, between cells, cells and the ECM, and the ECM itself determine the subsequent cell response. A high cytoskeletal tension causes differentiation, whereas a low tension maintains the undifferentiated state.8 Inspired by the ECM, many systems, both natural and synthetic, are developed for the culture of various cells in vitro, and as scaffolds for guiding regeneration in vivo.

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3 Inspired by these complex structures present in the ECM, biomaterials have been developed for regenerative medicine applications. These biomaterials can be categorized into 2D systems and elastomers useful as load bearing scaffolds in vivo, and 3D hydrogels which can be used for the culture of therapeutically relevant numbers of cells in vitro, and can be used as a model system for studying development, disease progression, and drug efficacy testing (figure 1).

Figure 1: A cartoon of the ECM as an inspiration for the design of an artificial ECM mimic for regenerative

medicine applications. Supramolecular elastomers serve as load bearing materials in vivo, whereas supramolecular hydrogels for the in vitro culture of cells.

1.2 2D-polymer systems

One of the most important properties in biomaterials design is the macroscopic stiffness of the material. Much effort has been dedicated towards directing stem cell differentiation by tuning the stiffness of the 2D cellular microenvironment.9 Mesenchymal stem cells typically differentiate into neurons when seeded on soft gels, whereas osteoblasts are formed on stiff gels. Moreover, the cell becomes stiffer during differentiation as was observed by a difference in response to external forces.10, 11 In vivo, every tissue has its own distinct elastic modulus, which is important in maintaining the homeostasis of the cell.12 However, when the matrix composition changes and the deposition increases, stiffness levels can increase by 10-fold, indicating cancer formation. In tumor progression, typically an elevated concentration of collagen type I and fibronectin is observed.13 By changing the stiffness of the cellular surroundings, it has also been shown that lineage specification can be redirected and differentiated cells can be reprogrammed into induced pluripotent stem cells.14, 15

Next to controlling the stiffness of the cellular microenvironment, epitope presentation has been extensively studied by researchers as well, in order to investigate the cell-ECM interactions.16 Typically the spacing of the integrin binding peptide ligand RGD is tightly controlled on microarrays. A spacing of 70 nm has been defined as a density threshold value in which a higher density spacing (< 70 nm) results in focal adhesions and the formation of cytoskeletal stress fibers, whereas a lower density fails to support cell adhesion and causes

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apoptosis.17 Moreover, it is assumed that cells are capable of sensing adhesion molecules within 5-10 nm distance with respect to their plasma membrane.18

1.3 Elastomers

Although 2D materials fail to recapitulate the 3D in vivo situation, they can be considered extremely valuable as load bearing scaffolds for tissue replacements.19, 20 Elastomers are polymers formed in organic media and deposited into a scaffold by e.g. electrospinning, solvent casting or 3D printing. Due to their dense and bundled structure, they typically exhibit a higher elastic modulus as compared to 3D hydrogels. The pores can be designed in such a way that cells can infiltrate or remain on the surface.

For tissue engineering, scaffolds can be seeded with cells upon which they could be implanted in vivo.21 However, this approach requires in vitro expansion of sufficient amounts of cells, which is challenging due to a quick loss of phenotype upon cell isolation, and culturing in natural or synthetic hydrogels prior to seeding. Typically, upon transplantation, most of the cells undergo apoptosis due to a change in cellular environment and nutrient depletion, and might induce an immune reaction. Another more successful approach is to implant an acellular inert scaffold in vivo, i.e. in situ tissue engineering. This allows immediate transplantation and uses the patient itself as a bioreactor. Cells are recruited to adhere to the scaffold, and are promoted to secrete ECM material. In time, more ECM is deposited which should be direct proportional with the degradation of the scaffold, eventually yielding into newly formed functional tissue. This strategy is successfully utilized in heart-valve replacement.22

Despite its success it is rather difficult to isolate cells from the scaffold on demand, which is required for the in vitro expansion of cells. Moreover, the microporous structure is typically too rigid for most cells and stem cells to maintain their phenotype and the pores are either too small and stable restricting cell growth into bigger structures, or too big, which yields in a more 2D environment than a 3D. Therefore, hydrogels are proposed as better candidates for the in vitro expansion of cells and stem cells to arrive at therapeutically relevant amounts of cells in vitro for cell modeling and regenerative medicine applications.

1.4 3D-hydrogel systems

Hydrogels form an attractive class of biomaterials since their aqueous environment can mimic the ECM. The hydrophilic polymers are able to absorb up to 99% of water, allowing for the encapsulation of cells under physiological conditions. Next to the water molecules bound to the polymer chains, free water molecules filling up the space in the network are able to allow for nutrient diffusion.23 This provides a supportive 3D environment closely resembling the in vivo situation. Hydrogel networks are formed by crosslinking and/or physical chain entanglements, of which the crosslinking mechanism can be mainly categorized based on covalent or physical interactions. Covalent networks are generally

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5 permanent whereas physical networks are reversible. Hence, the polymer concentration and/or the number of crosslinks can be regulated to tune the macroscopic properties.

Since the ECM is highly dynamic, hydrogels based on reversible crosslinks better recapitulate the native environment.24, 25 Non-covalent supramolecular interactions are highly dynamic where the non-covalent bond is in equilibrium. Several types of supramolecular interactions exist, usually in the form of directional hydrogen bonds, electrostatic interactions, π-π interactions and hydrophobic effects. Networks can be formed where supramolecular moieties are presented along a polymer backbone, however, fiber-like nanostructures upon self-assembly of amphiphilic monomeric units closer mimic the fibrous structures found in nature in e.g. the basement membrane. Especially collagen type IV, which is known as the network forming collagen solely present in the basement membrane, is only a few nanometers in diameter due to the entanglement of only three peptide chains around each other, rather than bundled fibrils.3, 26 Also laminin (~2-7 nm) and fibronectin (~2 nm) consist of long peptide chains of only a few nanometers in diameter.27

In one-dimensional nanofibers formed by self-assembly in aqueous solutions, the amphiphilic character of the monomeric units is essential. The hydrophilic part allows for water solubility, whereas the non-covalent interactions should be protected from water penetration. Tight packing, due to stronger hydrophobic effects and/or hydrogen bonding results in stiffer macroscopic material properties, whereas disordered packing results in softer hydrogels due to increased water penetration. Therefore the internal dynamics and macroscopic physical properties of supramolecular hydrogels can be tuned by varying the packing of the monomers within the fiber.28, 29

The ECM provides a niche to the embedded cell, regulating the behavior and the survival of cells. Inspired by this phenomena, naturally derived or synthetic ECM mimics provide an excellent support for the culture of cells and stem cells. These platforms play important roles in e.g. elucidating the role of specific compounds on cell behavior, directing the differentiation of stem cells into specific cell lineages, and up-scaling of specific cells or multi-cellular structures for regenerative medicine purposes.30, 31 In particular, organoids, which are in vitro cultured multi-cellular structures from pluripotent or adult stem cells, are becoming increasingly important in the biomedical field since they closely recapitulate the

in vivo organs and can possibly be used to regenerate diseased tissue.32, 33 Hence these

organoid platforms can be used to obtain valuable insights into tissue development, homeostasis and disease progression. So far, the culture of organoids is optimized in animal derived culture media, like Matrigel (which is explained in the natural hydrogels section), which gives rise to organoids which are variable in size and viability, therefore lacking the reproducibility properties of in vivo organs. By using biomaterials, a more precise control of the environment can be obtained which might lead to more consistent organoid structures.

With the purpose to culture stem cells in a 3D environment for biomedical applications, it is important that the hydrogel meets certain design criteria. Here, we propose a few design criteria that are considered important for the design of hydrogels, where the key factors of the ECM, ECM-cell interactions, physical properties and growth factor presentation - are included:

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 Cells should be encapsulated under physiological conditions.

 Control of the physical properties of the hydrogel is important to match the in vivo situation.

 Cell adhesion sites should be incorporated in the hydrogel.

 The hydrogel should allow dynamics, like degradation, remodeling, capturing of secreted ECM components.

 High cell viability and long-term expansion should be supported as well as the maintenance of a specific phenotype.

 The hydrogel should be able to capture, stabilize and release tissue specific growth factors.

Next to these criteria, the synthetic hydrogels should be available at large scale for a reasonable price.

1.4.1 Natural hydrogels

The naturally derived hydrogel most frequently used for the expansion of stem cells is Matrigel. Matrigel contains extracted extracellular matrix proteins from Englebreth-Holm Swarm sarcoma cells which provides an optimal and highly biofunctional natural environment for stem cells, hence maintaining the self-renewal and pluripotency of the embedded stem cells.34, 35 Despite being very useful for the long term expansion of stem cells, Matrigel has several disadvantages: 1) the composition is highly heterogeneous, with a batch-to-batch similarity of only 50-60%, causing reproducibility problems; 2) it is extracted from tumor cells preventing clinical use and 3) the stiffness of the hydrogel is extremely soft which cannot be tuned to match the tissue of origin.

To overcome the last two challenges, tissues have been isolated and decellularized to obtain tissue specific ECM. Upon removal of the cells, the shape, structure and components can be maintained and have shown promising results in vitro and in vivo.36 However, decellularization methods can damage the composition, giving rise to dysfunction or even a loss of important ECM components. Since the composition is hard to control, synthetic alternatives are proposed to provide a better solution.

1.4.2 Synthetic covalent hydrogels

Many covalent hydrogels are developed that show great promise for the 3D culture of stem cells. Compared to many research performed on 2D substrates, where the physical properties and epitope presentation direct cell lineage differentiation and spreading behavior, in 3D systems, the micro-environmental cues have a significant different impact on the cell response.37 To better recapitulate the in vivo situation, a 3D environment where the cell should be able to remodel its environment is required. Both the groups of Mooney

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and Chen 39 showed that the traction forces applied by the cells are able to reorganize the RGD ligands into clusters, which is not possible on extremely rigid substrates. Although the crosslink density can be tuned to match the mechanical properties of the in vivo situation, a too high crosslink density was shown to restrict cell spreading, proliferation and 3D migration.40, 41 In order to address this problem, Lutolf and coworkers incorporated

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7 enzymatic cleavable crosslinks in their design to enable local degradation of the cell to the surrounding environment. This proved to be highly successful since cell spreading and migration was promoted again compared to stiff non-degradable hydrogels.41 Next to enzymatic degradation of hydrogels, also hydrolytic degradable hydrogels were employed to control the degree of degradation, for example by Burdick and coworkers.42 Moreover, it is important to notice that there exists a delicate balance between degradation and cell growth; fast degradation may result in a loss of cell support, whereas a slow degradation might inhibit cell growth due to compressive forces. In addition, altered biomaterial properties upon different stages of cell growth might be important. This was beautifully demonstrated by Lutolf et al. where a relatively stiff environment with fibronectin was required for organoid proliferation, whereas soft mechanical properties and laminin functionality was necessary to drive organoid expansion.43 Finally, we would like to highlight the importance of growth factor sequestering and stabilization to protect growth factors from denaturation. Maynard and coworkers screened several covalent polymers on their ability to stabilize proteins, showing the importance of installing sulfonated, zwitterionic or trehalose side chains on polymers.44, 45 By immobilizing growth factors using electrostatic interactions, significant lower amounts of growth factors are required, and a prolonged presentation to growth factor receptors is achieved.

The lessons learned from covalent systems indicate that there is an increasing need for reversible and adaptive (i.e. dynamic) systems, allowing clustering of epitopes and remodeling of the ECM, local degradation of the hydrogel to allow expansion and/or migration, and shielding growth factors from inactivation yet presenting them to the cell. Supramolecular hydrogels have the advantage over covalent systems that they are assembled from small monomeric building blocks held together by weak non-covalent interactions. Due to the transient non-covalent bonds between monomers (i.e. equilibrium of associated and dissociated bonds), the monomers are able to diffuse in and between fibers. Therefore, supramolecular structures are intrinsically dynamic, hence responsive and adaptive, and the requirement for small monomeric building blocks enables modularity and tunability.24Since fibrous structures closer resemble the ECM environment, here a focus on hydrogel systems formed by the assembly of fibrous structures is provided.

1.4.3 From covalent to supramolecular hydrogels

In order to allow local adaptable properties yet maintaining long-term stability of a hydrogel, several hybrid systems have been developed.46 Incorporation of reversible bonds requires the use of non-covalent interactions or dynamic covalent interactions. In dynamic covalent chemistry, covalent bonds can be temporarily broken and reformed again.47 Although interesting self-healing materials were developed under physiological conditions,48 reversibility is usually slower than non-covalent interactions and might require the addition of a catalyst. Moreover, when crosslinking kinetics are too slow, cellular adhesion might be hampered, which results in sedimentation of cells to the bottom of the culture plate causing unfavorable 2D growth instead of 3D. Non-covalent moieties have been attached to covalent polymers to induce crosslinking by directional host-guest interactions, or by introducing secondary interactions by hydrophobic, hydrogen bonding or electrostatic interactions. For example, Scherman et al. are using host-guest interactions based on

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cucurbit[n]uril,49 and have recently developed a non-fibrous dual network with a trace amount of covalent crosslinks forming stretchable and though hydrogels.50 Moreover, Rowan and coworkers developed strain stiffening polymers by having a polyisocyanide backbone enriched with reversible hydrogen bonds between the amino acids in the side chains.51 Although the hybrid hydrogels bring along highly stretchable materials with though mechanical properties, these systems lack the ability to reposition functional epitopes along a fibrous backbone, in order to enable clustering and optimal integrin spacing as compared to fully non-covalently assembled systems. Only a few examples exist that are able to form nanofibrous structures capable of repositioning functional epitopes, such as self-assembling peptides, peptide amphiphiles and supramolecular amphiphiles. More details and excellent reviews about peptide based supramolecular hydrogels are found elsewhere (e.g. 52-54).

1.4.4 Self-assembling peptides

Self-assembling peptides have the advantage over synthetic polymers that they are intrinsically dynamic and naturally derived, which provides them with biodegradable and non-toxic properties. Self-assembling peptides are mainly ionically complementary. This means that by alternating positive and negative charges, the peptides can be stacked. The most frequently used self-assembling peptide is RADA16-I,55 commercialized as PuraMatrix. This peptide self-assembles in stable β-sheets via electrostatic interactions between the positively charged arginine (R) and negatively charged aspartic acid (D), with additional hydrophobic interactions between the alanine units (A) (figure 2a and 2b). Fibers are formed with a diameter of about 10 nm (figure 2c) and have been investigated in several in vitro and in vivo studies. Recently, Chen and coworkers attached a brain derived neurotrophic factor (BDNF) peptide onto RADA16 and showed that the co-culture of human umbilical cord mesenchymal stem cells and activated astrocytes promoted the proliferation and differentiation of neural cells in lesions after traumatic brain injury (figure 2d).56 Unfortunately, apoptosis was observed in larger, about 5 mm diameter, injured cavities. Neuroregeneration was also observed when induced pluripotent stem cells were encapsulated in RADA16-I scaffolds and after neuronal induction transplantation in a mouse brain.57 When the cells were encapsulated after neural induction, the survival in vitro was only limited. Despite major steps are made in in vivo studies, long term viability of cells remains a concern.

The peptide P11-4 (Ac-QQRFEWEFEQQ-NH2), commercialized as CurodontTM,

self-assembles into β-sheets containing fibrous structures, depending on the concentration and pH, using a combination of electrostatic interactions between the arginine and glutamic acid, hydrophobic interactions between the aromatic rings and hydrogen bonding between glutamines.58 The anionic groups of the glutamic acids were proposed to attract calcium ions, inducing mineralization in dental caries-like lesions.59 In 2013, P11-4 was shown to repair early dental lesions in patients by inducing remineralization 30 days post-treatment,60 and currently, this peptide completed phase 2 clinical trials.61 Although promising results were obtained and the way to clinical trials is paved, the structures used here can be further optimized to accelerate remineralization. Since only a small amount of negative charges was included in the system, the system might benefit from including proteoglycan mimics or phosphorylated groups.

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9 Figure 2: The self-assembling peptide RADA16-I. A) Chemical structure, B) schematic representation of

stacking, C) AFM (tapping mode) measurement showing fibers (scale bar indicates 100 nm). Modified from 55, PLoS One. D) Brain regeneration in a 2 mm lesion cavity when seeding activated astrocytes and Human umbilical cord mesenchymal stem cells embedded in a BDNF functionalized RADA16 hydrogel (left) or when injecting only saline (right) after 1-8 weeks. Adapted and modified from 56 with permission from Elsevier.

Nanofibrous structures based on Fmoc protected dipeptides have also been developed and have been shown to support the culture of chondrocytes in 2D and 3D environments.62 Self-assembly is induced by a combination of hydrogen bonding and π-π interactions, and upon using a pH switch. Self-supporting hydrogels were observed at <1 wt% concentration with a fiber diameter in the range of 19-68 nm. Chemical functionality, i.e. amine, carboxylic acid and hydroxyl, were incorporated at the end of the dipeptides and were shown to assemble into 1D fibers as well, however the mechanical properties at the hydrogel level were significantly influenced.63 Although the tested gels supported the culture of bovine chondrocytes, only the hydroxyl terminated peptide nanofibers supported other cell lines. Dalby et al. investigated the influence of hydrogel stiffness and the incorporation of metabolites into Fmoc-peptide nanofibers on the differentiation of perivascular stem cells.64 It was shown that specific lipids were consumed during the differentiation into chondrocytes and osteocytes, which demonstrates the importance of metabolites in directing the differentiation into a specific cell lineage. Yet, incorporating functional epitopes, like cell adhesive peptides, might further enhance cell viability.

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1.4.5 Self-assembling peptide amphiphiles

Peptide amphiphiles (PA) consist of a peptide part and a long hydrophobic tail which initiates hydrophobic collapse into cylindrical micelles (figure 3). Several functional amino acids as well as adhesive peptides have been incorporated in PAs to introduce biofunctionality, and were shown to form 1D fibers despite the chemical modifications.65 The fibronectin derived peptide RGDS was attached to the periphery of the monomers and was shown to induce the adhesion of several cells and stem cells.66 In contrast to covalent networks, where the spacing of the RGD needs to be tightly regulated17 or multivalent RGD display is required,67 in supramolecular fibers, bioactive cues can migrate along the fiber backbone allowing adaptation and optimal integrin spacing. Stupp and coworkers demonstrated that a significantly lower amount of bioactive guest presentation is required (i.e. about 5 mol%) to achieve cell attachment corroborating this hypothesis.68 The main question is whether these highly dynamic systems are able to resist the pulling forces applied by the cells. In principle, the non-covalent interactions that induce supramolecular polymer formation are relatively weak and can be broken. However, due to multiple hydrogen bonds, hydrophobic effects and additional β-sheet or π-π stacking, the packing of these structures can be regulated. For example, Stupp et al. also showed that disordered fiber packing can be obtained by substituting valine for alanine. As a consequence, β-sheet formation was decreased and disordered, which reduced the physical properties of the gel at the macroscopic level.29

Figure 3: An example of a self-assembled peptide amphiphile. A) Chemical structure, B) cartoon illustration of

assembly. Adapted and modified from 69 with permission from Elsevier. C) STEM image of co-assembled glucose-PA and carboxylic acid-PA, D) MSC encapsulated in Glucose functionalized PA co-assembled with glutamic acid end functionalized PAs (bottom) showed articular cartilage regeneration within 12 weeks after transplantation as compared to saline (top) and Hyalagan (middle). Adapted and modified from 70 with permission from the American Chemical Society.

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11 By introducing a glucose functionalized amino acid in PAs, Guler and coworkers aimed to mimic hyaluronic acid by combining the glucose-PA with carboxylic acid terminated PAs (figure 3c and 3d).70 It was shown that mesenchymal stem cells differentiated into chondrocytes without the use of exogenous growth factors by targeting the CD44 receptor. Chondrogenic differentiation of MSCs was also studied by Tekinay and coworkers by utilizing a heparin mimicking PA.71 By co-assembling a sulfonate functionalized PA, a carboxylate and an amine end-functionalized PA, the importance of epitope presentation on chondrogenesis was demonstrated. Although promising results are obtained, functional epitopes on peptide amphiphiles are closely displayed to the fiber backbone, thereby inducing steric hindrance of the backbone when binding to a target. Spacing of functional cues on RADA16-I was elucidated by investigating both the self-assembly behavior and the epitope presentation. It was demonstrated that 4 glycines improved both the stability and the presentation.72 An improved cell spreading was also observed when 5 glycines were used to space RGD epitopes on PAs.73 While a longer spacer length decreases steric effects of the backbone improving epitope availability, a spacer which is too long might have a decreased binding strength due to gained flexibility.

1.4.6 Supramolecular self-assembling amphiphiles

Although peptides are non-toxic and biodegradable, peptide synthesis on large scale is quite costly. Therefore, there is an increasing need for cheap, large scale alternatives. As a consequence, assembling amphiphilic monomers are required which have a self-assembling core, and a water soluble periphery.

The self-complementary UPy molecule was modified with PEG to enable water solubility, a urea moiety to allow lateral hydrogen bonding, and hydrophobic linkers to protect the inner hydrogen bonding core from water (figure 4).74 A combination of hydrophobic effects, hydrogen bonding and π-π stacking enables the self-assembly of these monomeric units into 1D fibers. Moreover, telechelic (bivalent) UPy molecules spaced by different lengths of PEG were developed and were shown to form hydrogels above a critical concentration. By changing the hydrophobic-to-hydrophilic ratio of the molecule, different fiber lengths were observed, and a difference in monomer exchange was elucidated.28 By varying the packing of the monomers in the fiber, functional epitopes can be presented in a highly dynamic fashion, or frozen in the backbone. A few examples exist where UPy hydrogels were shown to be useful in biomedical applications. For example, UPy hydrogels have been loaded with growth factors by physical entanglement and have been shown to reduce scar collagen in a myocardial infarction pig model (figure 4d).75 However, fast release of growth factors was observed, which can be improved by including functional epitopes in the fibers. By incorporating growth factor binding peptides into supramolecular fibers, growth factors can be captured via electrostatic interactions and released in a more sustained fashion. Although other functional UPy hydrogels have been developed,76 the culture of stem cells has not been reported yet.

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Figure 4: An example of a UPy based self-assembling amphiphiles. A) Chemical structure, top; bivalent UPy

(n=20kDa), bottom; functional UPy monomer, B) schematic representation of fiber formation, C) cryo-TEM of UPy based fibers (scale bar represents 100 nm). Adapted from 28 – published by the Royal Society of Chemistry. D) cardiac repair upon treatment with a pristine UPy hydrogel (left), growth factors HGF and IGF-1 in saline (middle) or physical entangled growth factors in UPy hydrogel (right). Scar tissue (white stain) was reduced, 4 weeks after treatment with growth factor loaded UPy-PEG hydrogel. Adapted from 75 with permission from Wiley.

Figure 5: BTA based self-assembled amphiphiles. A) Chemical structure, left; ethylene glycol BTA, right;

functional BTA monomer. B) schematic representation of BTA stack formation. Adapted from 77 with permission from the American Chemical Society. C) cryo-TEM of ethylene glycol based BTA fibers (scale bar represents 100 nm). Adapted from 78 – published by the Royal Society of Chemistry.

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13 Benzene-1,3,5-tricarboxamide (BTA) based molecules have also been modified to allow water solubility and were shown to form fibers in water due to a combination of 3-fold hydrogen bonding, a hydrophobic pocket protecting the inner core from water penetration and π-π interactions between the benzene rings (figure 5).78 A subtle change in the length of the aliphatic spacer was shown to have a tremendous effect on the internal dynamics in the fiber, also showing the importance of the hydrophobic-to-hydrophilic ratio.79 Similar to the UPy, BTA based hydrogels were formed at higher concentrations, and crosslinking was induced by introducing telechelic BTAs.80 Functional BTA hydrogels were developed where positively charged BTAs were incorporated in the fibers and were shown to capture siRNA by electrostatic interactions (figure 5B).77 While these systems are highly investigated in their structural and dynamic properties, they need to be investigated in vitro to examine their suitability for the culture of cells and stem cells.

1.5 Aim of the thesis

Inspired by the extracellular matrix, different hydrogel designs are being developed which are evaluated on their potential use for the 3D culture of cells and organoids. Synthetic hydrogels are required as substitutes of current culture environments because of the ill-defined, heterogeneous and highly complex composition with a batch-to-batch variation of animal-derived hydrogels. In particular supramolecular hydrogels show great promise, since their fibrous morphology is able to recapitulate dynamic processes found in nature which might support the expansion of cells in a 3D fashion due to its adaptable properties. Due to the defined and animal-free cell environment, a more accurate fundamental understanding of tissue development, homeostasis and disease progression can be obtained. Moreover, improving the culture of cells and organoids allows the ability to arrive at therapeutically relevant amounts of cells for the regeneration of diseased tissue. Although the latter is still elusive, small steps are being made. When designing biomaterials it is important to understand the physical and biochemical properties of the material in great detail, since optimizing the biomaterial to meet the desired cell response requires controlled modifications on the biomaterial.

The research described in this thesis elaborates on the design and evaluation of supramolecular biomaterials for their potential use in the expansion of organoids for regenerative medicine applications. Herein three main aims are distinguished to address the most critical issues in evaluating a potential biomaterial. The first objective is to obtain a

fundamental understanding of the internal fiber dynamics and assembly properties of UPy

and BTA based supramolecular polymers in solution. To integrate biofunctionality into the supramolecular polymers by incorporating various active peptides on the periphery, and to evaluate their specific function is the second aim. The last goal is to perform in vitro

experiments for the expansion of organoids using two organoid types in a fully synthetic

environment to assess their translational potential. The experiments performed in this thesis open up opportunities for supramolecular based hydrogels for the culture of Matrigel-free organoids for transplantation purposes.

In Chapter 2 two UPy based monomers are compared, monovalent UPy 1 and bivalent UPy 2, on their structural properties and their monomer exchange upon self-assembly. A

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small percentage of dye-labeled monomers, either Cy3 (green) or Cy5 (red), is incorporated in the UPy-based fibers and the monomer exchange is evaluated upon mixing using Förster resonance energy transfer (FRET) and stochastic optical reconstruction microscopy (STORM). Although both monomers form fibrous self-assembled structures, a large difference in monomer exchange rates is observed. Monovalent UPy 1 is shown to self-assemble into tightly packed 1D fibers exhibiting slow monomer exchange, whereas bivalent UPy 2 displays a fast exchange probably due to disordered self-assembly caused by the long ethylene glycol linker.

In Chapter 3, a system to investigate cell-supramolecular polymer interactions is proposed. By staining the cell membrane with a FRET acceptor and functionalizing supramolecular polymers with its donor at the end of the cell adhesive moiety RGD, the stability of functionalized monomers in different UPy based materials could be elucidated. Initial experiments are performed showing a difference in FRET upon using different UPy-based scaffolds. However, the system still needs to be further optimized and tested to investigate the fate of the functionalized monomers in time.

In chapter 4 sulfated and sulfonated peptides are investigated on their ability to stabilize heparin binding growth factors in solution. The sulfonated peptide SP3 is shown to improve the stability of transforming growth factor β1 (TGF-β1), and upon incorporation in a supramolecular system a synergistic stabilization effect is observed. High cellular activity is observed in cell experiments indicating that the growth factor remains highly active upon binding to the excipients. The stabilization of bone morphogenetic protein 4 (BMP4) is also elucidated highlighting the applicability of sulfonated functionalized supramolecular polymers for the stabilization of various heparin binding growth factors.

Supramolecular saccharide-based BTAs are introduced in chapter 5, which can be used as the second component in assembling a dual network multi-component biomaterial. The self-assembly and co-assembly of glucose and cellobiose functionalized BTAs is investigated in detail using several techniques. BTA-glucose is shown to assemble into long helical fibers, whereas BTA-cellobiose is unable to do so. In contrast, when co-assembling BTA-cellobiose with either BTA-glucose or ethylene glycol BTA, similar absorption and scattering is observed as the BTA-glucose and ethylene glycol BTA indicating fibrous assembly.

Chapter 6 describes the installation of antimicrobial activity in two different systems.

Choline binding peptides (CBPs) are coupled onto BTAs and dendrimers. Unfortunately, BTA-CBP incorporated in ethylene glycol BTAs are proved to be unstable in buffer hampering their use in antimicrobial experiments. Although the degree of CBP functionalization on the G2 and G3 dendrimer is not defined, antimicrobial activity is significantly improved upon higher degree of multivalency, i.e. G3 as compared to G2 and CBP.

The fundamental knowledge obtained in previous chapters is used to build a laminin mimicking UPy hydrogel in chapter 7, in order to support the expansion of intestinal organoids. Different organoid encapsulation strategies are elucidated showing diverse cellular behavior. Design criteria are proposed for an improved design of supramolecular hydrogels as biomaterials for the expansion of intestinal organoids.

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15 In chapter 8 the expansion of liver organoids in UPy-based supramolecular hydrogels is explored. A significant higher organoid viability is observed as compared to the intestinal organoids investigated in chapter 7. Both the morphology and gene expression levels are evaluated showing liver functionality. This opens up opportunities to further evaluate the material as a culture scaffold to arrive at therapeutically relevant numbers of organoids.

Chapter 9evaluates the aims stated above with reference to the research performed in

this thesis. A perspective on the future of organoid expansion for in vitro and in vivo applications is provided as well as the design of the next generation of supramolecular biomaterials is discussed. Bottlenecks of the current design and their possible solution(s) are proposed. In order to bring supramolecular biomaterials to the next level for clinical applications, high-throughput screenings, bioreactors, and multi-component biomaterials are requested.

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Chapter 2

TUNING THE DYNAMIC NATURE OF

SUPRAMOLECULAR POLYMERS

The structural properties of biomaterials are known to have an enormous influence on cell behavior. Although little is known about the effect of internal dynamics of supramolecular polymers on cells in vitro, it is very likely that dynamic non-covalent interactions will initiate different cell responses than rigid covalent polymers due to increased remodeling capabilities. In this chapter, the structural and kinetic exchange properties of supramolecular polymers composed of mono- and bivalent ureidopyrimidinone-based monomers are investigated in aqueous solutions. By using mono- and bivalent UPy-based monomers functionalized with a dye, it was shown that the internal dynamics is regulated by the scaffold type and to a lesser extent by the valency of the dye. Moreover, the exchange dynamics can be controlled by mixing different types of monomers. This tunability can be used in the design of biomaterials in order to match the desired cell response in vitro.

Part of this work has been published:

S.I.S. Hendrikse, S.P.W. Wijnands, R.P.M. Lafleur, M.J. Pouderoijen, H.M. Janssen, P.Y.W. Dankers and E.W. Meijer, Controlling and tuning the dynamic nature of supramolecular polymers in aqueous solutions, Chem. Commun., 2017, 53, 2279-2282.

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2.1 Introduction

Water-soluble supramolecular polymers constitute an attractive class of polymers, since the non-covalent interactions between the molecular components can result in tunable hydrogels with a highly dynamic structure.1-3 When applied as biomaterial, the dynamic, adaptable, and reversible interactions between the hydrogel and embedded (stem) cells are proposed to closely mimic systems and processes found in nature. The artificial biomaterial developed shall not only contain specific mechanical properties, but also has to present proteins and peptides to cells in a reversible, adaptive and spatiotemporalmanner in order to regulate cell response.4, 5 Therefore, control over the structural and dynamic properties of hydrogels is important for the design and development of supramolecular biomaterials, which can only be achieved when there is profound understanding at the molecular level.

Many techniques are currently used to study such supramolecular systems in aqueous solutions.6 Fӧrster resonance energy transfer (FRET) experiments7, 8 have been used to study

exchange dynamics in a broad spectrum of systems from liposomes9 to polymers. In

addition, fluorescence microscopy, and more recently super resolution microscopy techniques such as stochastic optical reconstruction microscopy (STORM) yield valuable structural and temporal information of systems ranging from giant unilamellar vesicles to 1D-supramolecular aggregates such as benzene-1,3,5-tricarboxamide based polymers and peptide amphiphiles.10-12 Furthermore, stimulated emission depletion (STED) microscopy

has been successfully applied in studying the formation and characteristics of multicomponent self-sorted supramolecular hydrogels in real-time.13

Using molecular design parameters to tune macroscopic properties of hydrogels has been nicely shown by Scherman and Stupp. Scherman has shown that different guests for cucurbit[8]uril mediated crosslinking resulted in distinct crosslink dynamics, influencing the mechanical properties at the hydrogel level.14 Stupp has revealed that a disordered fiber packing can be obtained by substituting valine for alanine in peptide amphiphiles, which subsequently decreases the physical properties of the hydrogel.15

In this chapter, the structural and kinetic properties at the nanometer scale of supramolecular ureidopyrimidinone (UPy) based polymers in water are investigated. The UPy moiety is a self-complementary unit containing a quadruple hydrogen bonding array. In chloroform the high binding constant of 6*107 M-1 results in a bonding life-time of roughly one second.16 In order to obtain similar values in water, the UPy unit requires hydrophobic shielding from the water-soluble oligo- and poly(ethylene glycol) groups.17 Additionally, urea groups are introduced in the hydrophobic alkyl spacers to enable lateral stacking into long 1D-polymers due to a combination of hydrophobic effects, π-π stacking and hydrogen bonding. Recently, it has been shown that both mono- and bivalent UPy monomers assemble into supramolecular polymers that give hydrogels at concentrations of only a few weight percentage (wt%).17 By mixing the mono- and bivalent UPy monomers, hydrogels are obtained of which the stiffness can be controlled by the composition of the mixture.18

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2.2 Results and Discussion

Defining structural differences between the scaffolds. The structural and temporal

properties of the assembly of monovalent monomers mUPy 1 and bivalent monomers bUPy

2 (figure 1) in water have been elucidated using cryogenic transmission electron microscopy

(cryo-TEM), STORM and FRET experiments. For the latter two techniques, 2 mol% of dye-labelled probes (monovalent mDye 3a, mDye 3b, and bivalent bDye 4a, bDye 4b; figure 1) were embedded in the aggregates formed by the monomers 1 and 2. For the synthesis of these labelled probes the reader is referred to the experimental section.

Figure 1: Molecular structures of monovalent mUPy 1, bivalent bUPy 2, dye-labelled monovalent UPy mDye 3a

and mDye 3b, and dye-labelled bivalent UPy bDye 4a and bDye 4b. The scaffolding materials 1 and 2 are studied in combination with 2 mol% of dyes 3 and 4 that serve as molecular probes. The UPy based molecules dimerize via quadruple hydrogen bonding arrays (grey) and laterally stack via the urea moieties (red). The poly(ethylene glycol) spacers (blue) provide water solubility.

Solutions of 1 and 2 in water were stirred at 72 °C for 1.5 hours, and were then slowly cooled to room temperature. Subsequently, the structural characteristics of the scaffolds formed by 1 and 2 were compared using cryo-TEM. Micrometers long fibrillar structures with diameters of approximately 5 and 14 nm were observed for 1 (figure 2A and B), while shorter fibrillar structures in the range of hundreds of nanometers with an apparent diameter of approximately 7 nm were found for 2 (figure 2D and E). The 14 nm diameter of

1 corresponds to bundled dimers, whereas the 5 nm of 1 and 7 nm of 2 are proposed to

correspond to single dimers. Please note that the water shell surrounding the PEG part of the molecules does not absorb sufficient electrons. In the case of 2, the longer PEG surrounding the fiber backbone sequesters more water which blurred the contrast of the inner hydrophobic part. The incorporation of the dye-labelled monomers 3a, 3b, 4a, and 4b within these fibers was investigated using STORM. The measurements were performed at

N NH O N H NH H N HN N H O O O O O 3 6 O 20kDa NH H N HN N H O O NH 6 3 O N HN O N NH O N H NH H N HN N H O O O O O 3 6 O 10 O O O N H 11 O O O N N H O O N H O O O O 11 11 5 NN HN N O N O O N N N NH O N H NH H N HN N H O O 3 6 N NH O N H NH H N HN N H O O 3 6 6 O 3 O N H H N HN N H NH O HN N 1 2 3a 4a O O O N H 11 O N N NH O N H NH H N HN N H O O 3 6 3b N O O N N H O O N H O O O O 11 11 5 NN HN N O O O N NH O N H NH H N HN N H O O 3 6 6 O 3 O N H H N HN N H NH O HN N 4b N Cl Cl Cl Cl

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12.5 μM sample concentrations, using 1 or 2 in combination with 2 mol% of the dye-labelled probes 3a, 3b, 4a, or 4b. Micrometers long fiber scaffolds were observed for 1 (with mDye

3b, figure 2C), corroborating with the cryo-TEM results for 1. In contrast, smaller,

less-defined 1D-aggregates were observed for 2 (with mDye 3b, figure 2F) with a maximal length of a few hundreds of nanometers. Despite the >20 fold poorer spatial resolution,19 the ~40 times lower concentration and/or effects of sample preparation and adsorption on the glass surface, these structures formed by 2 appeared relatively similar as compared to the features observed with cryo-TEM. Nevertheless, the results of the two techniques showed a clear structural difference between the aggregates formed by the two UPy derivatives, where the long fibers of 1 and the shorter and less-defined fibers of 2 indicate a differentiation in packing of the monomers.

Figure 2: Cryo-TEM and STORM images of scaffolds formed by monovalent mUPy 1 (A-C) and bivalent bUPy 2

(D-F). A) and D) are detailed cryo-TEM images (scale bare represents 100 (A) and 200 nm(D)); B) and E) are cryo-TEM overview images (scale bar indicates 0.5 (B) and 1 µm (E)); C) and F) are STORM images (scale bar represents 1 µm). Fibrillar structures were observed in both mUPy 1 and bUPy 2 with a length of a few micrometers and diameter of approximately 5 and 14 nm for mUPy 1 (c = 0.5 mg/ml; 482 µM) and a length of a few hundreds of nanometers and a diameter of approximately 7 nm for bUPy 2 (c = 10 mg/ml; 474 µM). STORM images indicate successful incorporation of mDye 3b (cTotal=12.5 µM, c3b=0.25 µM).

Probing the energy exchange of dye-functionalized monomers in different scaffolds. Next,

the excellent FRET pair, donor Cy3 and acceptor Cy5,20 was selected for studying the exchange of monomeric units in and between 1D-fiber scaffolds. Separate solutions containing Cy3 (mDye 3a or bDye 4a) and Cy5 (mDye 3b or bDye 4b) were prepared by incorporating 2 mol% of these dye-probes 3a, 3b, 4a or 4b into pre-assembled scaffold solutions containing 1 or 2. Specifically, 24.5 µM scaffold, and 0.5 µM dye probe concentrations were used (sample preparation described in experimental section). Subsequently, the corresponding Cy3 and Cy5 solutions (i.e. 3a and 3b, or 4a and 4b) were

A B C

(30)

23 Figure 3: A) Emission scans recorded after mixing solutions of mDye 3a and 3b or bDye 4a and 4b incorporated

in scaffolds formed by mUPy 1 or bUPy 2 at different time points (cScaffold=24.5 µM, cDye=0.5 µM, excitation at

540 nm). Immediate exchange occurred in samples containing bUPy 2 whereas very low FRET was observed in samples containing mUPy 1. B) Co-assembly of mUPy 1 with bUPy 2, using 2 mol% of mDyes 3a+3b or bDyes

4a+4b incorporated (cTotal=25 µM). The normalized FRET intensity was obtained by plotting the normalized

intensity Cy5 peak upon Cy3 excitation. Upon increasing bUPy 2 content, the exchange dynamics was increased due to a disordering of the monomer packing. The bDyes 4a+4b displayed slower energy transfer as a result of improved anchoring as compared to mDyes 3a+3b in the same fiber composition.

mixed in a 1:1 ratio. Upon excitation of the donor, the fluorescence of both dyes was measured (figure 3A). It is known that close proximity (2-10 nm) of both dyes results in efficient energy transfer.8 Because of the non-covalent interactions between the monomers,

the dye-labelled monomers can migrate within and between fibers. As a result, this technique investigates the exchange dynamics of the dye-labelled monomers within and

mDye3a+3b (0.5 µM) bDye4a+4b (0.5 µM)

bU P y 1 (24 .5 µM ) m U P y 2 (24 .5 µM ) m D ye 3a +3 b (0 .5 µM ) bD ye 4a +4 b (0 .5 µM ) Co-assembly (25 µM) A B

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