INSEGURIDAD JURÍDICA EN CONTRATACIÓN ESTATAL

Supramolecular interactions were utilized to produce physical hydrogels that evolved through hierarchical assembly to produce highly porous microstructures over time. Assembly resulted from macromolecular condensation, driven by dynamic molecular guest-host complexation. Micromechanical analysis indicated that the pores were indeed devoid of solid hydrogel—containing only low concentrations of dissociated polymer from stochastic erosion. Furthermore, the porosity evolved temporally to increase both void fraction and pore diameter. Owing to polymer solvation and electrostatic repulsion, component concentration was primarily responsible for suppression of the void fraction. Reduction in network dynamics (i.e., increased polymer modification) influenced the timescale of macromolecular rearrangement, altering pore diameter. Through these mechanisms, pore diameters spanning three orders of magnitude were achieved and void fractions as great as 93.3±2.4% were observed. These studies begin to close the gap in knowledge which exists between directed polymeric assembly and microstructure within supramolecular hydrogels.

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Complexation with Cucurbit[8]uril. J. Am. Chem. Soc. 132, 14251-14260 (2010).

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28. Mealy, J. E., Rodell, C. B. & Burdick, J. A. Sustained small molecule delivery from injectable hyaluronic acid hydrogels through host–guest mediated retention. J. Mat. Chem. B3, 8010-8019 (2015).

29. Appel, E. A., Loh, X. J., Jones, S. T., Dreiss, C. A. & Scherman, O. A. Sustained release of proteins from high water content supramolecular polymer hydrogels. Biomaterials33, 4646-4652 (2012).

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Stem Cell Therapy. Adv. Healthc. Mater.4, 237-244 (2015).

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CHAPTER 6

Selective Proteolytic Degradation of Guest-Host Assembled,

Injectable Hyaluronic Acid Hydrogels

Adapted from: Rodell CB, Wade RW, Purcell BP, Dusaj NN, Burdick JA. Selective Proteolytic Degradation of Guest-Host Assembled, Injectable Hyaluronic Acid Hydrogels. ACS Biomaterials Science & Engineering 1, 227-237 (2015).

6.1 Introduction

Hydrogels are well suited for a range of biomedical applications, particularly due to their biophysical and biochemical properties. While synthetic hydrogels are not typically bioactive, they may be regarded as a blank slate, where desirable signals (e.g., topography, adhesion, mode of degradation) can be introduced. The incorporation of these signals has been widely used to engineer synthetic extracellular matrix (ECM) analogues for use as therapeutic scaffolds for tissue engineering or drug delivery applications. Such an engineering approach allows for reproducibility and more accurate control over physiochemical properties, when compared to naturally derived materials that exhibit many of the same features.1,2

The incorporation of degradation into hydrogels is necessary for many applications, such as to control therapeutic delivery or to enable autonomous, noninvasive clearance of implanted or injected materials. Although numerous

degradation mechanisms exist (e.g. hydrolysis,2-5 _{and stochastic surface erosion}6- 8_{), recent studies have focused on incorporating enzymatically degradable peptide}

crosslinkers9 _{to engineer cell-material interactions,}10-14 _{drug delivery systems,}1,15- 17 _{tissue engineering scaffolds,}18-21 _{and biosensors.}22-24 _{Such enzymatic}

degradation may be accomplished through polymeric or oligopeptide based crosslinkers with sensitivity to various enzymes, as discussed in numerous reviews.2,4,25,26 _{Of these enzymatically responsive systems, degradation by matrix}

metalloproteinases (MMPs)14,27,28 _{is particularly powerful, owing to the array of}

known MMPs, their specific expression patterns by cells and in response to injury,29 _{and the tailorable enzyme-substrate activity.}30,31 _{These features lend}

themselves to therapeutic use, such as the generation of specific degradation in response to cell invasion or tissue damage.

For employment of such systems in vivo, an implantable solid hydrogel may be formed by crosslinking ex vivo. This approach has been quite useful, including in drug delivery applications where cell-mediated degradation results in controlled biomolecule release.28,32 _{Alternatively, these systems may be injectable, such as}

through Michael-addition crosslinking where MMP-mediated delivery of multiple growth factors covalently bound to hydrogels is possible.16 _{Hydrogel formation}

through Schiff-base addition reactions has also enabled in situ hydrogel formation, with degradation used to modulate release of an MMP inhibitor, constituting a feedback system for modulation of tissue remodeling processes.17 _{While such}

covalent chemistries may enable injection, they pose the potential clinical challenge of premature crosslinking and delivery failure. These concerns are

particularly evident in the case of percutaneous delivery, where materials may be necessarily held within the injection device (i.e., syringe or catheter tubing) for a prolonged period (>1 hr) prior to injection.

To address such challenges, self-assembling hydrogels may be of great utility. These systems may be exemplified by supramolecular self-assembling hydrogels, formed through the specific interaction of pendant or end group functionalities. In the majority of cases, these noncovalent interactions are readily reversible, enabling shear-induced flow for injection and rapid recovery at the target site for high retention.33,34 _{Systems based on various guest-host chemistries}8,35 _or

engineered peptide and protein complexation7,36,37 _{demonstrate these desirable}

injectable properties. Numerous other noncovalently crosslinked networks have been formed that exhibit proteolytic degradation, including those based on thermogellation14,27 _{and ionic crosslinking.}19 _{Self-assembling peptide systems}

have also been reported,15,38-43 _{including where degradation behavior influences}

molecule release15 _{and encapsulated cell behavior.}39,43 _{While such systems show}

promise, their utility may be hampered by the limited scale of production and mechanical robustness, as well as difficulties associated with the synthesis and characterization of peptide units modified to incorporate degradable domains.40

Herein, we sought to harness the advantages of both shear-thinning injectable materials and protease degradation. To accomplish this, we employed the guest- host hydrogel system (Figure 6.1A), in which hydrogels undergo noncovalent hydrogel formation through the supramolecular interaction of adamantane (guest, Ad) and β-cyclodextrin (host, CD), which were separately coupled to hyaluronic

acid (HA). Such modification enabled formation of shear-thinning hydrogels with stochastically governed surface erosion. To permit proteolytic degradation, adamantane was bound via a peptide tether, which included the MMP degradable sequence VPMS-MRGG (Figure 6.1B, left) or the modified sequence VDMS- MAGG (Figure 6.1B, right).

Figure 6.1. Overview of material design. (A) Schematic of supramolecular assembly, with dynamic crosslink formation generated via guest-host complexation of β-cyclodextrin (host, CD) and adamantane (guest, Ad). (B) Design of the positively charged peptide linker (PAd – to allow MMP degradation) and negatively charged peptide linker (NAd – to limit specific MMP degradation), including Ad end-modification (i), cysteine residue for Michael- addition coupling (ii), MMP cleavage site (iii), and local variation of charge near the cleavage site as underlined in amino acid sequence (iv).