Chemical and Biological Engineering Strategies to Make and Modify Next-Generation Hydrogel Biomaterials

Abstract

There is a growing interest in the development of technologies to probe and direct in vitro cellular function for fundamental organoid and stem cell biology, functional tissue and metabolic engineering, and biotherapeutic formulation. Recapitulating many critical aspects of the native cellular niche, hydrogel biomaterials have proven to be a defining platform technology in this space, catapulting biological investigation from traditional two-dimensional (2D) culture into the 3D world. Seeking to better emulate the dynamic heterogeneity characteristic of all living tissues, global efforts over the last several years have centered around upgrading hydrogel design from relatively simple and static architectures into stimuli-responsive and spatiotemporally evolvable niches. Towards this end, advances from traditionally disparate fields including bioorthogonal click chemistry, chemoenzymatic synthesis, and DNA nanotechnology have been co-opted and integrated to construct 4D-tunable systems that undergo preprogrammed functional changes in response to user-defined inputs. In this Review, we highlight how advances in synthetic, semisynthetic, and bio-based chemistries have played a critical role in the triggered creation and customization of next-generation hydrogel biomaterials. We also chart how these advances stand to energize the translational pipeline of hydrogels from bench to market and close with an outlook on outstanding opportunities and challenges that lay ahead.

Keywords: Biofunctional; Biomaterials; Bioresponsive; Drug Delivery; Hydrogels; Synthetic Biology; Tissue Engineering.

Figure 1.. Key Milestones in the Deployment of New Chemistries for Hydrogel Biomaterial Synthesis

Key milestones marking the first instance of the deployment of a particular chemistry for the synthesis of a hydrogel biomaterial.

Figure 2.. Chain-Growth vs. Step-Growth Chemistries for Hydrogel Network Synthesis

(A) Radical-initiated chain-growth crosslinking results in a molecularly undefined and spatially heterogenous network. (B) Click chemistry-mediated step-growth networks spontaneously form more homogenous and well-defined mesh structures, typically without the need for initiators.

Figure 3.. Hydrogel Synthesis through SpyTag-SpyCatcher Ligation and Split GFP Reconstitution

(A) SpyCatcher and SpyTag react spontaneously upon mixing and form an isopeptide bond between Lys31 on SpyCatcher and Asp117 on SpyTag. (B) Star-like proteins bearing reactive 4 reactive SpyCatchers physically assemble through spontaneous reconstitution of split GFP. Adapted with permission from Yang et al. Copyright 2020, Elsevier. (C) Covalent crosslinking of 4-arm star-like protein macromers with difunctional SpyTag reagents yields a “Spy-G” hydrogel. Reproduced with permission from Yang et al. Copyright 2020, Elsevier.

Figure 4.. Assembly of Non-Covalently Linked Hydrogel Networks through Host-Guest Chemistry and Hydrophobic-Hydrophobic Interactions

(A) Host-guest chemistries enable the formation of physically and reversibly crosslinked hydrogels. (B) Demonstration of self-healing properties of a host-guest crosslinked hyaluronic acid-based hydrogel. White and red regions represent cyclic deformation at 0.5% and 250%, respectively. Storage and loss modulus are recovered after each cycle. Reproduced with permission from Rodell et al. Copyright 2013, The American Chemical Society. (C) Gel fragments formed by hydrophobic association undergo physical grafting when placed in direct contact. Reproduced with permission from Tuncaboylu et al. Copyright 2011, The American Chemical Society.

Figure 5.. Injectable and Printable Recombinant Protein Hydrogels

(A) Shear-thinning behavior of the physical network and superior biocompatibility of many recombinant protein-based hydrogels make them attractive targets for injectable therapies and extrusion-based bioprinting applications. As these materials are pushed through narrow passages, increased shear stress cause reversible liquefication, carrying along any cellular or biochemical cargo. (B) A burst-resistant bi-layer patch uses two engineered variants of a shear-thinning leucine zipper-based hydrogel. The inner layer imitates the mechanical properties of softer native heart tissue, while the outer layer provides structural stability. Burst resistance of the was modulated by recombinant introduction of mussel foot protein domains Mefp3 and Mefp5 into the leucine zipper crosslinker, generating a chimeric set with an array of mechanical properties. Adapted with permission from Jiang et al. Copyright 2022 Wiley-VCH. (C) Recombinant protein hydrogels uniquely allow iterative and high-throughput screening of physicochemical and biological properties through classic and next-generation protein engineering techniques. Plots adapted with permission from Dooling and Tirrell. Copyright 2016 The American Chemical Society.

Figure 6:. Engineering Dynamic Biomaterials through Aptamer Biology

(A) When incorporated into a hydrogel backbone, aptamers in an extended initial state can lead to macroscopic-level changes in network mechanical properties through basic biorecognition and cognate target capture. (B) Layered hydrogels are synthesized such that the top hydrogel (fluorescent green) is crosslinked through ATP-binding extended state aptamers and the bottom hydrogel is crosslinked through insulin-binding extended state aptamers. When exposed to the appropriate cognate molecule (ATP in the top and insulin in the bottom network), conformational changes in the aptamer crosslink lead to significant network volume decrease. Image reproduced with permission from Bae et al. Copyright 2018, Wiley-CVH. (C) Aptamers can be engineered as force-mediated release systems. Traction Force Activated Payloads (TrAPs) are designed such that an aptamer bound to a target molecule is also linked to an RGD motif that recognizes force-responsive integrin motifs. Upon local mechanosensing or application of a force stimulus, unfolding of the aptamer leads to target molecule release. (D) TrAPs enable selective activation of growth factors in 3D collagen scaffolds by Primary Human Smooth Muscle Cells. Extent of release and variation between different cell types is due to the relative expression of different adhesion receptors. Images for (C) and (D) reproduced with permission from Stejskalova et al. Copyright 2019, Wiley-CVH.

Figure 7.. Aptamer-Enabled Targeting and Capture of Circulating Tumor Cells

A) Aptamer-initiator bi-block constructs are designed to specifically bind to epithelial cell adhesion molecules (epCAMs) which are highly expressed on the membranes of tumor cells. Following binding, biblocks containing an initiator trigger the formation of an encapsulating DNA hydrogel. Post-encapsulation, ATP can be used to effect a conformational change within the ATP-responsive aptamer to destroy the gel, leading to tumor cell release. B) H1 and H2 – which are step-look-structured – are in a metastable state because of the protective effects of long stems in their secondary structures. In the presence of the initiator, the hybridization reaction is triggered leading to hydrogel assembly. C) Confocal microscopic imaging showing aptamer-initiator biblock binding to the cell surface membrane. Scale bar: 10 μm. D) Multilayered cells can be found encapsulated within the DNA hydrogel when stained with FDA dyes. Stack height: 40 μm. E) Cells disperse in solution upon ATP-triggered release. Scale bar: 100 μm. Image reproduced with permission from Ye et al. Copyright 2020, Springer Nature.

Figure 8.. Sortase-mediated Gel Functionalization and Multimaterial Degradation

(A) Sortase selectively ligates polyglycine-tagged cargo onto LPXTG-containing peptide sequences covalently bound to the polymer network. (B) Harnessing evolved sortases’ ability to recognize orthogonal peptide motifs found within crosslinkers comprising different gel regions, staged material degradation and accompanying cellular release can be achieved; for example, with eSrtA(4S9), then eSrtA(2A9), then eSrtA-5M. Adapted with permission from Bretherton et al. Copyright 2023, Wiley-VCH. (C) Sequential sortase treatment enables user-defined control over cell-laden multimaterial degradation. Maximum intensity projections of the University of Washington logo, comprised of cells constitutively expressing one of three fluorescent proteins, are shown prior to degradation (left), following treatment with eSrtA(4S9) treatment (center), and following eSrtA(2A9) treatment (right). Scale bars = 1 mm. Reproduced with permission from Bretherton et al. Copyright 2023, Wiley-VCH.

Figure 9.. Design and Synthesis of Cas-Reponsive Hydrogel Networks

Methacryl-functionalized DNA is incorporated into polyacrylamide chains (PA-X, PA-Y) during starting macromer polymerization. This enables different routes to gel actuation and response modes. In one example, shown on the left, the Cas12a–gRNA is added to the gel precursor with the nanoparticle cargo, before the addition of dsDNA cues and ssDNA crosslinker. In another example, shown on the right, cell encapsulation is triggered through the addition of a small amount of ssDNA bridge crosslinker to the macromers mixed in solution. This thickens the pre-gel solution and minimizes losses incurred during the washing step. More ssDNA linker is then added at the same time as the cells to fully crosslink the hydrogels. Finally, the experiment is initiated by exposing the gels to gRNA-complexed Cas12a and dsDNA. Additional details of the crosslinking strategy (bottom of the panel): the two ends of the DNA bridge hybridize with distinct ssDNA anchors incorporated into polyacrylamide macromers, while the central AT-rich portion remains single-stranded and sensitive to Cas12a collateral activity. Reproduced with permission from Gayet et al. Copyright 2020, Springer Nature.

Figure 10.. User-Engineered and Directed Biomaterial Responsiveness

(A) Material inputs such as light-, enzyme-, and reductant-responsiveness can be codified as Boolean logic crosslinkers. Adapted with permission from Badeau et al. Copyright 2018, the authors. (B) Biological epitopes can be photo-patterned with pristine spatiotemporal control in order to recapitulate native physiological structures ex vivo (reaction platform shown here is a photomediated oxime ligation). (C) Three-dimensional patterning of an anatomical heart is achieved in a fibrin-based hydrogel network through photomediated oxime ligation, showcased with 3D and cross-sectional cut views (mCherry-CHO is shown in red). (Scale bar: 50 μm). (D) Hydrogel networks can be engineered to reversibly photostiffen/photosoften through judicious engineering of a secondary photolabile linker. (E) Photodegradation of hydrogel networks can yield shapes and geometries with pristinely conserved features such as endothelialized 3D vascular networks. Adapted with permission from Arakawa et al. Copyright 2020, the authors.