Controlled spatial patterning of ligand presentation within hydrogels.

State of the art

Natively, cells reside in a complex microenvironment, called the extracellular matrix (ECM), where they receive numerous signals (chemical, mechanical, biological) that allow them to maintain proper function; however, cells can be damaged and these interactions disrupted as a result of injury or disease. To address this problem, tissue engineering is developing as an emerging field with the ambitious aim to combine biological and engineering-based approaches to restore damaged tissue function1. To create an appropriate cell-instructive environment for implanted cells or regenerating tissues, an as yet unachieved goal is to develop a fully synthetic matrix that recapitulates the complex spatial and temporal patterning of bioactive molecules within native tissue architecture using elegant chemical approaches that are both simple to implement and compatible with many different signaling molecules. Synthetic materials systems address many of the concerns, such as manufacturing, reproducibility, and immunogenicity, associated with biologically derived molecules. Excitingly, recent and increasing discoveries of short bioactive peptides, such as RGD2 (single letter amino acid abbreviations used throughout), as well as the existence of solid-phase synthesis techniques make possible the concept of peptide-functionalized materials and thus fully synthetic bioactive materials. However, the ‘minimal’ complexity of signal presentation needed to lead to a robust functional response remains an unanswered and important question. In particular, the relative spatial distribution of these signals is neglected within existing materials systems. And yet, appropriate spacing is needed, for example, for synergistic ligand signaling. These ligand pairs have fixed separations within natural proteins, and currently fusion protein and protein domain production remain as standard approaches for their related co-presentation3,4.

In the design of synthetic materials incorporating biological functionality, hydrogels have excellent potential as cell encapsulation matrices because they can mimic key properties of native tissue in a controlled way. Hence, hydrogels can serve as a stimulating environment that can drive encapsulated cells to a functional tissue state, and recent research has led to many novel developments5. Key advances include the engineering of cell-responsive poly(ethylene glycol) (PEG) hydrogels, which present cell adhesive signals and can be enzymatically degraded because of the incorporation of protease-sensitive substrates within the hydrogel network6. Increasingly complex spatially patterned or temporally dynamic hydrogels have allowed the presentation of multiple signals, such as by the inclusion of stimulus-sensitive linkers to permit photo-patterning of channels after the cell-encapsulated gel has been formed7. Patterning of RGD on hydrogel surfaces has also been used to create regions that promote cell attachment on an otherwise unfavorable substrate8. Further, enhancement of the PEG hydrogel system has been performed to allow increased enzymatic remodeling rates through the incorporation of optimized protease-sensitive substrates9,10 and to promote self-renewal of encapsulated stem cells through the co-presentation of multiple cell-adhesive signals within one hydrogel11.

Scientific Goals

This CREA proposal addresses the goal of creating fully synthetic ECM mimetics by implementing facile chemical approaches to achieve complex patterning of bioactive molecules. The new idea behind the project is that one can control the relative spatial positions of two (or more) peptides within a hydrogel in a modular approach, that is, without affecting other gel properties. As the influence of spatial control of molecule presentation has not been properly explored because the technology is lacking (what this proposal will now solve), it could pave the way to the clinical application of tissue engineered products by addressing important drawbacks of biologic-based drugs, such as lack of specificity, high doses required, and uncontrolled side effects. To provide biological signals to cells growing on or in an engineered matrix, a generic strategy is to expose them to proteins, protein domains3,4, or even short bioactive peptides2. While many peptides or proteins have proven biological effects, the specific spatial presentation of these molecules is crucial for their proper function and their full activity. For example, as described above, the spacing between synergistic ligands is often established by their fixed positions within a protein3,4. However, most biomimetic scaffolds present biomolecules homogeneously distributed in the bulk of the material. One interesting class of materials that may serve as a potential tool for controlling the spatial presentation of bioactive molecules includes self-assembling amphiphilic peptides, which have themselves been developed as hydrogel-forming materials12. These self-assembling peptide amphiphiles can be functionalized with bioactive molecules13,14; however, there is always a concern that such modifications will interfere with gel network formation. Other self-assembling materials have been rendered bioactive, as recently reviewed15, providing a means for the modular incorporation of a variety of molecules within one construct. However, these approaches do not address the relative spatial orientation of the incorporated molecules.

Thus, the goal of this project is to demonstrate feasibility that incorporation of self-assembling domains within PEG hydrogels provides a straightforward chemical reaction scheme can be used to create matrices that present varied bioactive molecules with controlled nano-scale organization as well as the potential for additional spatial patterning at the micro- or macro-scale. While it may seem logical to combine the PEG hydrogel and self-assembling peptide technologies described above, it is not obviously evident that this approach will work. Therefore, the first aim of this project is to develop a novel hybrid hydrogel system to allow spatially controlled clustered display of pendant bioactive peptides on self-assembling tethers that are covalently bound to multi-arm PEG macromers. Specifically, PEG formulations that form stable hydrogel networks6,9 will be functionalized with peptides possessing self-assembling capability, such as RADA-1612, to promote clustering of bioactive ligands within the hydrogel. Then, to demonstrate that these matrices are not only bioactive but can also result in enhanced cellular function, the second aim is to use this system to address questions relevant to micro-environmental influence on cell signaling. Clustered presentation of a single ligand will be used to determine optimal parameters, such as the number of peptides per cluster and the overall concentration of ligand, to enhance cell adhesion and migration. If successful, the further clustering of synergy sequences will be examined for increased receptor signaling and resulting effects on cell adhesion and migration.

Research Method

In the context of creating technologies to enable the development of fully synthetic ECM mimetics, the overall objective of this two-year CREA project is to utilize molecular self-assembly approaches for the spatially controlled clustered presentation of cell-adhesive signals within hydrogels. To achieve this objective, two aims are envisioned. The first aim is to demonstrate the proof of principle that self-assembling ‘tethers’ can be incorporated into PEG hydrogels for the spatially controlled clustered display of pendant molecules. The second aim is to use this hybrid hydrogel system to explore biological applications of the clustered presentation of bioactive peptides.

In the first aim, multi-arm PEG macromers, end-functionalized with vinyl sulfone (VS) groups, will be reacted with self-assembling peptide segments containing a free cysteine in a Michael-type addition reaction. An excess of PEG-VS will be added to the reaction to ensure only one peptide per PEG molecule; this is necessary to retain sufficient reactive groups to form a hydrogel network through Michael-type addition reactions with di-thiol containing compounds. Two approaches will be explored to promote clustering of pendant signals. In the first approach, the concentration of tethered peptide within the mixture will be varied to determine the critical concentration for self-assembly. In the second approach, additional "free" self-assembling peptide segments will be added to the mixture to promote assembly of the peptide domains. Ultimately, the second approach would allow for a greater concentration of peptide within the hydrogel system as well as allow incorporation of different bioactive peptides without the need to create new PEG-peptide macromers each time. Self-assembly will be monitored by fluorescence microscopy using tagged peptides. From a technical perspective, these experiments will be easy to perform as the synthesis of the peptide segments required is straight-forward and the Michael-type addition reactions can achieve high efficiency. Whether or not the peptide segments will self-assemble (the key step required to enable spatial control) when attached to PEG macromers remains a large unknown – this is likely the greatest risk factor for the project. However, in an opposite approach, self-assembling poly(alanine) domains from N. calvipes silk were combined with short-chain PEG segments to form self-assembling hydrogels16, which suggests that the presence of PEG in the mixture (at certain concentrations) should not interfere with peptide self-assembly. It will also be interesting to examine the effects of the incorporation of self-assembling segments on hydrogel microarchitecture. Peptide amphiphiles tend to form gels with a fibrillar network whereas PEG hydrogels are amorphous. It is possible that the presence of self-assembling domains within the PEG hydrogels may affect the microstructure of the gel (i.e., induce the formation of fibrillar regions) and thus its mechanical properties.

In the second aim, receptor-ligand interactions will be examined for different cell adhesion ligands and the integrins they bind. Integrins and their ligands are an excellent choice to study with the spatially controlled clustered presentation system designed in the first aim. For many integrins, short peptide sequences (3-10 amino acids) that bind to specific integrin subunits are known, which would facilitate peptide synthesis, and the presence of these peptide sequences in defined locations within ECM molecules points to the importance of the spatial orientation of their display. For example, the activity of the cell adhesion ligand, RGD, is enhanced by the co-presentation of its synergy sequence, PHSRN, in a neighboring domain within fibronectin3. Using RGD as a model system, readouts of biological activity will include receptor activation assays as well as functional assays of cell attachment or migration. Initially, experiments will be performed to demonstrate that clustered presentation of a single ligand can increase cell attachment or migration in both 2-D and 3-D hydrogel systems. For example, it already has been shown that the spatial distribution of RGD can affect cell adhesion and motility17. Adding complexity, we will then seek to demonstrate that clustered presentation of synergistic ligand pairs, such as RGD and PHSRN, can lead to enhanced signaling through their receptors and ultimately to enhanced cell function (spreading, migration, proliferation, differentiation, etc.). If the development of the hybrid hydrogels for clustered pendant peptide display proposed in the first aim is successful, the experiments proposed for the second aim represent just the beginning of the potential biological applications of this system. The ability to control spatial presentation of bioactive peptides would allow many interesting questions to be studied in the context of basic cell biology as well as have potential as a materials system for guiding cell behavior during in vitro tissue engineering or in vivo tissue regeneration.

The innovative aspect of this project includes the development of novel hydrogel materials, with a specific emphasis on the ability to control the relative spatial presentation of two or more peptide ligands – a technique that can also be implemented in 3-D. Additionally, it could lead to the creation of entirely synthetic approaches to explore receptor/ligand signaling, which would bypass many of the cumbersome molecular biology steps involved in fusion protein and protein domain production. Peptide ligands of any receptors with synergistic signaling can easily be incorporated in this system. Further, due to the cell-mediated enzymatic remodeling capacities that can be engineered into the PEG hydrogels, effects on both 2-D and 3-D cell adhesion, spreading, migration, and proliferation can be explored within one system. Development of this hybrid hydrogel system would also set the stage for future work by allowing the possibility of covalent or affinity binding of really any bioactive peptide into the matrix.