Researchers develop a safer and more precise chemical method for designing artificial environments that support cell growth and tissue engineering.
(Nanowerk Spotlight) In tissue engineering, replicating the environment that surrounds cells in the body remains one of the field’s central challenges. Hydrogels—water-rich polymer networks—are widely used to mimic this extracellular matrix. Their mechanical softness and high water content make them well-suited for hosting living cells.
Yet, many hydrogel systems rely on chemical reactions that inadvertently affect the cells they’re meant to support. These reactions can trigger unwanted side effects by interacting with biological molecules or creating harmful byproducts, especially when used to encapsulate sensitive cell types like stem cells.
This has prompted researchers to develop “bioorthogonal” chemistries—reactions that proceed under biological conditions without interfering with native cellular processes. Two widely adopted examples are strain-promoted azide-alkyne cycloaddition (SPAAC) and tetrazine-based inverse electron-demand Diels–Alder (IEDDA) reactions. Both enable cross-linking without toxic initiators or catalysts.
SPAAC is valued for its use of small, stable azides. IEDDA, while similarly specific, involves larger and more hydrophobic reactants that can affect the solubility and biocompatibility of hydrogel components. These limitations point to the need for additional bioorthogonal tools that offer fast, clean reactivity while maintaining compatibility with biological systems.
A study published in Advanced Functional Materials (“Bioorthogonal Engineering of Cellular Microenvironments Using Isonitrile Ligations”) introduces isonitrile-based ligation as an alternative approach to cross-linking hydrogels under physiological conditions. Isonitriles, also called isocyanides, are compact, chemically stable, and rarely found in nature. This makes them unlikely to interact with proteins, nucleic acids, or other cellular components. The research evaluates three isonitrile-compatible reactions: those involving chlorooximes (ChO), azomethine imines (AMI), and tetrazines (Tz). Each can form covalent bonds under aqueous conditions without requiring light, heat, or metal catalysts.
Among these, the isonitrile-ChO reaction showed the most practical features for hydrogel formation. When polyethylene glycol (PEG) chains functionalized with isonitrile groups were combined with PEG-ChO under neutral pH at room temperature, gelation occurred rapidly—within five minutes. Compared to the AMI and Tz reactions, which required elevated temperature or acidic conditions to proceed at a similar rate, isonitrile-ChO gelation offered a more convenient and biologically compatible setup.
Researchers compared three chemical reactions—isonitrile with chlorooximes, azomethine imines, and tetrazines—for forming hydrogels suitable for living cells. All three reactions successfully created gels, but the isonitrile-chlorooxime reaction worked fastest and required only mild conditions. This reaction also produced gels with stable structure, good elasticity, and no gas bubbles. Hydrogels made with this method remained intact over time in cell culture conditions and supported high cell survival. Encapsulated human cells showed over 95% viability, demonstrating that the materials are both stable and biologically safe. (Image: reprinted from DOI:10.1002/adfm.202422047, CC BY) (click on image to enlarge)
Mechanical testing showed that the resulting hydrogels were elastic and stable when incubated in cell culture conditions. However, increasing the reaction temperature led to faster gelation but also introduced structural defects, likely due to incomplete cross-linking or degradation of intermediates. This was confirmed by the presence of unreacted isonitrile groups and reduced gel stiffness. Optimal performance was achieved at 22 °C, balancing gelation speed with network integrity.
By adjusting the polymer concentration, researchers could fine-tune the stiffness of the hydrogel, covering a range relevant to various tissue types. Higher polymer content yielded stiffer gels and faster gelation. Frequency and strain sweep tests confirmed that the material maintained elastic behavior across physiologically relevant ranges.
The team then extended the system by incorporating engineered elastin-like proteins (ELPs), which allow the introduction of biological cues. These proteins were modified to display either RGD peptides—short sequences that promote cell attachment—or non-functional RDG controls. Isonitriles were introduced onto the ELPs through their lysine residues, enabling them to integrate seamlessly into the PEG-ChO network. This modular design made it possible to independently vary the gel’s cross-linking density and its biological signaling properties.
When human fibroblasts were encapsulated in these hybrid gels, their behavior reflected the underlying design. In networks with low cross-link density and abundant RGD sites, cells spread extensively and displayed elongated shapes. In contrast, cells in tightly cross-linked gels or in matrices presenting only RDG remained rounded. These results confirmed that both matrix remodelability and biochemical adhesion are required for cells to spread and migrate in three dimensions.
To test biocompatibility more broadly, the authors embedded several human cell types in the isonitrile-ChO hydrogels, including vascular endothelial cells, neural stem cells, and primary muscle progenitor cells. All maintained high viability after encapsulation and throughout extended culture. Endothelial cells formed networks of tube-like structures and retained expression of CD31, a vascular marker. Neural stem cells preserved stemness markers and differentiated into neurons and astrocytes under the right conditions. Muscle progenitor cells maintained their identity and were capable of forming striated myotubes following induction. This confirmed that the hydrogels support cell-specific function as well as survival.
A key strength of the isonitrile-ChO chemistry lies in its selectivity. The researchers demonstrated this by comparing isonitrile-based hydrogel functionalization to the more commonly used thiol-maleimide reaction. When fluorescent dyes were introduced into pre-formed gels, the isonitrile ligation reacted only with the hydrogel matrix and not with cellular proteins. In contrast, the maleimide-thiol reaction labeled both the gel and the encapsulated cells, showing unwanted cross-reactivity. Moreover, maleimide-based gels degraded over time, while isonitrile-ChO gels remained intact, reinforcing their suitability for long-term culture.
The researchers also tested whether isonitrile ligation could operate in parallel with SPAAC. They synthesized ELPs that contained both isonitrile and azide groups and showed that each could be selectively targeted with its corresponding dye. The reactions were mutually orthogonal and remained active after a week in culture, enabling precise dual modifications to the same gel. This capability opens the door to multistage control over the chemical environment surrounding cells—such as delivering one factor early in culture and another later to guide differentiation or morphogenesis.
The isonitrile-ChO reaction meets the critical benchmarks for constructing functional hydrogel networks. It proceeds quickly and under mild conditions, avoids toxic byproducts, and enables fine control over material architecture. The resulting gels maintain structural stability, resist degradation in serum-rich media, and support both cell survival and function across a variety of human cell types. Their compatibility with other bioorthogonal chemistries like SPAAC adds an additional layer of flexibility for applications that require temporal or spatial control over the cell environment.
The findings position isonitrile ligations as a practical addition to the bioorthogonal chemistry toolbox. Their selectivity, modularity, and compatibility with live cells and other chemistries make them well-suited for future applications in regenerative medicine, therapeutic delivery, and engineered tissue systems.
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