Reversible Intracellular Gelation of MCF10A Cells Enables Programmable Control Over 3D Spheroid Growth.

In nature, some organisms survive extreme environments by inducing a biostatic state wherein cellular contents are effectively vitrified. Recently, a synthetic biostatic state in mammalian cells is achieved via intracellular network formation using bio-orthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) reactions between functionalized poly(ethylene glycol) (PEG) macromers. In this work, the effects of intracellular network formation on a 3D epithelial MCF10A spheroid model are explored. Macromer-transfected cells are encapsulated in Matrigel, and spheroid area is reduced by ≈50% compared to controls. The intracellular hydrogel network increases the quiescent cell population, as indicated by increased p21 expression. Additionally, bioenergetics (ATP/ADP ratio) and functional metabolic rates are reduced. To enable reversibility of the biostasis effect, a photosensitive nitrobenzyl-containing macromer is incorporated into the PEG network, allowing for light-induced degradation. Following light exposure, cell state, and proliferation return to control levels, while SPAAC-treated spheroids without light exposure (i.e., containing intact intracellular networks) remain smaller and less proliferative through this same period. These results demonstrate that photodegradable intracellular hydrogels can induce a reversible slow-growing state in 3D spheroid culture.

Photo-expansion microscopy enables super-resolution imaging of cells embedded in 3D hydrogels.

Hydrogels are extensively used as tunable, biomimetic three-dimensional cell culture matrices, but optically deep, high-resolution images are often difficult to obtain, limiting nanoscale quantification of cell-matrix interactions and outside-in signalling. Here we present photopolymerized hydrogels for expansion microscopy that enable optical clearance and tunable ×4.6-6.7 homogeneous expansion of not only monolayer cell cultures and tissue sections, but cells embedded within hydrogels. The photopolymerized hydrogels for expansion microscopy formulation relies on a rapid photoinitiated thiol/acrylate mixed-mode polymerization that is not inhibited by oxygen and decouples monomer diffusion from polymerization, which is particularly beneficial when expanding cells embedded within hydrogels. Using this technology, we visualize human mesenchymal stem cells and their interactions with nascently deposited proteins at <120 nm resolution when cultured in proteolytically degradable synthetic polyethylene glycol hydrogels. Results support the notion that focal adhesion maturation requires cellular fibronectin deposition; nuclear deformation precedes cellular spreading; and human mesenchymal stem cells display cell-surface metalloproteinases for matrix remodelling.

Intracellular Crowding by Bio-Orthogonal Hydrogel Formation Induces Reversible Molecular Stasis.

To survive extreme conditions, certain animals enter a reversible protective stasis through vitrification of the cytosol by polymeric molecules such as proteins and polysaccharides. In this work, synthetic gelation of the cytosol in living cells is used to induce reversible molecular stasis. Through the sequential lipofectamine-mediated transfection of complementary poly(ethylene glycol) macromers into mammalian cells, intracellular crosslinking occurs through bio-orthogonal strain-promoted azide-alkyne cycloaddition click reactions. This achieves efficient polymer uptake with minimal cell death (99% viable). Intracellular crosslinking decreases DNA replication and protein synthesis, and increases the quiescent population by 2.5-fold. Real-time tracking of single cells containing intracellular crosslinked polymers identifies increases in intermitotic time (15 h vs 19 h) and decreases in motility (30 µm h-1  vs 15 µm h-1 ). The cytosol viscosity increases threefold after intracellular crosslinking and results in disordered cytoskeletal structure in addition to the disruption of cellular coordination in a scratch assay. By incorporating photodegradable nitrobenzyl moieties into the polymer backbone, the effects of intracellular crosslinking are reversed upon exposure to light, thereby restoring proliferation (80% phospho-Rb+ cells), protein translation, and migration. Reversible intracellular crosslinking provides a novel method for dynamic manipulation of intracellular mechanics, altering essential processes that determine cellular function.

Using Stereochemistry to Control Mechanical Properties in Thiol-Yne Click-Hydrogels.

The stereochemistry of polymers has a profound impact on their mechanical properties. While this has been observed in thermoplastics, studies on how stereochemistry affects the bulk properties of swollen networks, such as hydrogels, are limited. Typically, changing the stiffness of a hydrogel is achieved at the cost of changing another parameter, that in turn affects the physical properties of the material and ultimately influences the cellular response. Herein, we report that by manipulating the stereochemistry of a double bond, formed in situ during gelation, materials with diverse mechanical properties but comparable physical properties can be obtained. Click-hydrogels that possess a high % trans content are stiffer than their high % cis analogues by almost a factor of 3. Human mesenchymal stem cells acted as a substrate stiffness cell reporter demonstrating the potential of these platforms to study mechanotransduction without the influence of other external factors.

Photoclick Chemistry: A Bright Idea.

At its basic conceptualization, photoclick chemistry embodies a collection of click reactions that are performed via the application of light. The emergence of this concept has had diverse impact over a broad range of chemical and biological research due to the spatiotemporal control, high selectivity, and excellent product yields afforded by the combination of light and click chemistry. While the reactions designated as "photoclick" have many important features in common, each has its own particular combination of advantages and shortcomings. A more extensive realization of the potential of this chemistry requires a broader understanding of the physical and chemical characteristics of the specific reactions. This review discusses the features of the most frequently employed photoclick reactions reported in the literature: photomediated azide-alkyne cycloadditions, other 1,3-dipolarcycloadditions, Diels-Alder and inverse electron demand Diels-Alder additions, radical alternating addition chain transfer additions, and nucleophilic additions. Applications of these reactions in a variety of chemical syntheses, materials chemistry, and biological contexts are surveyed, with particular attention paid to the respective strengths and limitations of each reaction and how that reaction benefits from its combination with light. Finally, challenges to broader employment of these reactions are discussed, along with strategies and opportunities to mitigate such obstacles.

Charged Poly(N-isopropylacrylamide) Nanogels for the Stabilization of High Isoelectric Point Proteins.

Storage and transportation of protein therapeutics using refrigeration is a costly process; a reliable electrical supply is vital, expensive equipment is needed, and unique transportation is required. Reducing the reliance on the cold chain would enable low-cost transportation and storage of biologics, ultimately improving accessibility of this class of therapeutics to patients in remote locations. Herein, we report on the synthesis of charged poly(N-isopropylacrylamide) nanogels that efficiently adsorb a range of different proteins of varying isoelectric points and molecular weights (e.g., adsorption capacity (Q) = 4.7 ± 0.2 mg/mg at 6 mg/mL initial IgG concentration), provide protection from external environmental factors (i.e., temperature), and subsequently release the proteins in an efficient manner (e.g., 100 ± 1% at 2 mg/mL initial IgG concentration). Both cationic and anionic nanogels were synthesized and selectively chosen based on the ability to form electrostatic interactions with adsorbed proteins (e.g., cationic nanogels adsorb low isoelectric point proteins whereas anionic nanogels adsorb high isoelectric point proteins). The nanogel-protein complex formed upon adsorption increases the stabilization of the protein's tertiary structure, providing protection against denaturation at elevated temperatures (e.g., 84 ± 4% of the protected IgG was stabilized when exposed to 65 °C). The addition of a high molar salt solution (e.g., 40 mM CaCl2 solution) to protein-laden nanogels disrupts the electrostatic interactions and collapses the nanogel, ultimately releasing the protein. The versatile materials utilized, in addition to the protein loading and release mechanisms described, provide a simple and efficient strategy to protect fragile biologics for their transport to remote areas without necessitating costly storage equipment.

Using Stereochemistry to Control Mechanical Properties in Thiol-Yne Click-Hydrogels.

The stereochemistry of polymers has a profound impact on their mechanical properties. While this has been observed in thermoplastics, studies on how stereochemistry affects the bulk properties of swollen networks, such as hydrogels, are limited. Typically, changing the stiffness of a hydrogel is achieved at the cost of changing another parameter, that in turn affects the physical properties of the material and ultimately influences the cellular response. Herein, we report that by manipulating the stereochemistry of a double bond, formed in situ during gelation, materials with diverse mechanical properties but comparable physical properties can be obtained. Click-hydrogels that possess a high % trans content are stiffer than their high % cis analogues by almost a factor of 3. Human mesenchymal stem cells acted as a substrate stiffness cell reporter demonstrating the potential of these platforms to study mechanotransduction without the influence of other external factors.

Calcium Signaling Regulates Valvular Interstitial Cell Alignment and Myofibroblast Activation in Fast-Relaxing Boronate Hydrogels.

The role viscoelasticity in fibrotic disease progression is an emerging area of interest. Here, a fast-relaxing hydrogel system is exploited to investigate potential crosstalk between calcium signaling and mechanotransduction. Poly(ethylene glycol) (PEG) hydrogels containing boronate and triazole crosslinkers are synthesized, with varying ratios of boronate to triazole crosslinks to systematically vary the extent of stress relaxation. Valvular interstitial cells (VICs) encapsulated in hydrogels with the highest levels of stress relaxation (90%) exhibit a spread morphology by day 1 and are highly aligned (80 ± 2%) by day 5. Key myofibroblast markers, including α-smooth muscle actin (αSMA) and collagen 1a1 (COL1A1), are significantly elevated. VIC myofibroblast activation decreases by 42 ± 18% through inhibition of mechanotransduction, independently of VIC morphology and alignment. Calcium signaling through a transient receptor potential vanilloid 4 (TRPV4) is found to regulate VIC spreading, alignment, and activation in a time dependent manner. Inhibition of calcium signaling at early time points results in disturbed cell alignment, decreased mechanotransduction, and diminished activation, while inhibition at later time points only causes partially reduced myofibroblast activation. These results suggest a potential crosstalk mechanism, where calcium signaling acts upstream of mechanosensing and can regulate VIC myofibroblast activation independently of mechanotransduction.

Viscoelasticity of hydrazone crosslinked poly(ethylene glycol) hydrogels directs chondrocyte morphology during mechanical deformation.

Chondrocyte deformation influences disease progression and tissue regeneration in load-bearing joints. In this work, we found that viscoelasticity of dynamic covalent crosslinks temporally modulates the biophysical transmission of physiologically relevant compressive strains to encapsulated chondrocytes. Chondrocytes in viscoelastic alky-hydrazone hydrogels demonstrated (91.4 ± 4.5%) recovery of native rounded morphologies during mechanical deformation, whereas primarily elastic benzyl-hydrazone hydrogels significantly limited morphological recovery (21.2 ± 1.4%).

Hydrogel and Organogel Formation by Hierarchical Self-Assembly of Cyclic Peptides Nanotubes.

Breaking away from the linear structure of previously reported peptide-based gelators, this study reports the first example of gel formation based on the use of cyclic peptides made of alternating d- and l-amino acids, known to self-assemble in solution to form long nanotubes. Herein, a library of cyclic peptides was systemically studied for their gelation properties in various solvents, uncovering key parameters driving both organogel and hydrogel formation. The hierarchical nature of the self-assembly process in water was characterised by a combination of electron microscopy imaging and small-angle X-ray scattering, revealing a porous network of entangled nanofibres composed by the aggregation of several cyclic peptide nanotubes. Rheology measurements then confirmed the formation of soft hydrogels.

Self-healing, stretchable and robust interpenetrating network hydrogels.

A self-healable stretchable hydrogel system that can be readily synthesized while also possessing robust compressive strength has immense potential for regenerative medicine. Herein, we have explored the addition of commercially available unfunctionalized polysaccharides as a route to synthesize self-healing, stretchable poly(ethylene glycol) (PEG) interpenetrating networks (IPNs) as extracellular matrix (ECM) mimics. The introduction of self-healing and stretchable properties has been achieved while maintaining the robust mechanical strength of the orginal, single network PEG-only hydrogels (ultimate compressive stress up to 2.4 MPa). This has been accomplished without the need for complicated and expensive functionalization of the natural polymers, enhancing the translational applicability of these new biomaterials.

Design of synthetic extracellular matrices for probing breast cancer cell growth using robust cyctocompatible nucleophilic thiol-yne addition chemistry.

Controlled, three-dimensional (3D) cell culture systems are of growing interest for both tissue regeneration and disease, including cancer, enabling hypothesis testing about the effects of microenvironment cues on a variety of cellular processes, including aspects of disease progression. In this work, we encapsulate and culture in three dimensions different cancer cell lines in a synthetic extracellular matrix (ECM), using mild and efficient chemistry. Specifically, harnessing the nucleophilic addition of thiols to activated alkynes, we have created hydrogel-based materials with multifunctional poly(ethylene glycol) (PEG) and select biomimetic peptides. These materials have definable, controlled mechanical properties (G' = 4-10 kPa) and enable facile incorporation of pendant peptides for cell adhesion, relevant for mimicking soft tissues, where polymer architecture allows tuning of matrix degradation. These matrices rapidly formed in the presence of sensitive breast cancer cells (MCF-7) for successful encapsulation with high cell viability, greatly improved relative to that observed with the more widely used radically-initiated thiol-ene crosslinking chemistry. Furthermore, controlled matrix degradation by both bulk and local mechanisms, ester hydrolysis of the polymer network and cell-driven enzymatic hydrolysis of cell-degradable peptide, allowed cell proliferation and the formation of cell clusters within these thiol-yne hydrogels. These studies demonstrate the importance of chemistry in ECM mimics and the potential thiol-yne chemistry has as a crosslinking reaction for the encapsulation and culture of cells, including those sensitive to radical crosslinking pathways.

Nonswelling Thiol-Yne Cross-Linked Hydrogel Materials as Cytocompatible Soft Tissue Scaffolds.

A key drawback of hydrogel materials for tissue engineering applications is their characteristic swelling response, which leads to a diminished mechanical performance. However, if a solution can be found to overcome such limitations, there is a wider application for these materials. Herein, we describe a simple and effective way to control the swelling and degradation rate of nucleophilic thiol-yne poly(ethylene glycol) (PEG) hydrogel networks using two straightforward routes: (1) using multiarm alkyne and thiol terminated PEG precursors or (2) introducing a thermoresponsive unit into the PEG network while maintaining their robust mechanical properties. In situ hydrogel materials were formed in under 10 min in PBS solution at pH 7.4 without the need for an external catalyst by using easily accessible precursors. Both pathways resulted in strong tunable hydrogel materials (compressive strength values up to 2.4 MPa) which could effectively encapsulate cells, thus highlighting their potential as soft tissue scaffolds.

Precision Epitaxy for Aqueous 1D and 2D Poly(ε-caprolactone) Assemblies.

The fabrication of monodisperse nanostructures of highly controlled size and morphology with spatially distinct functional regions is a current area of high interest in materials science. Achieving this control directly in a biologically relevant solvent, without affecting cell viability, opens the door to a wide range of biomedical applications, yet this remains a significant challenge. Herein, we report the preparation of biocompatible and biodegradable poly(ε-caprolactone) 1D (cylindrical) and 2D (platelet) micelles in water and alcoholic solvents via crystallization-driven self-assembly. Using epitaxial growth in an alcoholic solvent, we show exquisite control over the dimensions and dispersity of these nanostructures, allowing access to uniform morphologies and predictable dimensions based on the unimer-to-seed ratio. Furthermore, for the first time, we report epitaxial growth in aqueous solvent, achieving precise control over 1D nanostructures in water, an essential feature for any relevant biological application. Exploiting this further, a strong, biocompatible and fluorescent hydrogel was obtained as a result of living epitaxial growth in aqueous solvent and cell culture medium. MC3T3 and A549 cells were successfully encapsulated, demonstrating high viability (>95% after 4 days) in these novel hydrogel materials.

Efficient In Situ Nucleophilic Thiol-yne Click Chemistry for the Synthesis of Strong Hydrogel Materials with Tunable Properties.

Synthetic hydrogel materials offer the ability to tune the mechanical properties of the resultant networks by controlling the molecular structure of the polymer precursors. Herein, we demonstrate that the nucleophilic thiol-yne click reaction presents a highly efficient chemistry for forming robust high water content (ca. 90%) hydrogel materials with tunable stiffness and mechanical properties. Remarkably, optimization of the molecular weight and geometry of the poly(ethylene glycol) (PEG) precursors allows access to materials with compressive strength up to 2.4 MPa, which can be repeatedly compressed to >90% stress. Beyond this, we demonstrate the ability to access hydrogels with storage moduli ranging from 0.2 to 7 kPa. Moreover, we also demonstrate that by a simple precursor blending process, we can access intermediate stiffness across this range with minimal changes to the hydrogel structure. These characteristics present the nucleophilic thiol-yne addition as an excellent method for the preparation of hydrogels for use as versatile synthetic biomaterials.