Daniel L. Alge

Two-Dimensional Nanomaterials for Biomedical Applications: Emerging Trends and Future Prospects

Two-dimensional (2D) nanomaterials are ultrathin nanomaterials with a high degree of anisotropy and chemical functionality. Research on 2D nanomaterials is still in its infancy, with the majority of research focusing on elucidating unique material characteristics and few reports focusing on biomedical applications of 2D nanomaterials. Nevertheless, recent rapid advances in 2D nanomaterials have raised important and exciting questions about their interactions with biological moieties. 2D nanoparticles such as carbon-based 2D materials, silicate clays, transition metal dichalcogenides (TMDs), and transition metal oxides (TMOs) provide enhanced physical, chemical, and biological functionality owing to their uniform shapes, high surface-to-volume ratios, and surface charge. Here, we focus on state-of-the-art biomedical applications of 2D nanomaterials as well as recent developments that are shaping this emerging field. Specifically, we describe the unique characteristics that make 2D nanoparticles so valuable, as well as the biocompatibility framework that has been investigated so far. Finally, to both capture the growing trend of 2D nanomaterials for biomedical applications and to identify promising new research directions, we provide a critical evaluation of potential applications of recently developed 2D nanomaterials.

Synthetically tractable click hydrogels for three-dimensional cell culture formed using tetrazine-norbornene chemistry.

The implementation of bio-orthogonal click chemistries is a topic of growing importance in the field of biomaterials, as it is enabling the development of increasingly complex hydrogel materials capable of providing dynamic, cell-instructive microenvironments. Here, we introduce the tetrazine–norbornene inverse electron demand Diels–Alder reaction as a new cross-linking chemistry for the formation of cell laden hydrogels. The fast reaction rate and irreversible nature of this click reaction allowed for hydrogel formation within minutes when a multifunctional PEG-tetrazine macromer was reacted with a dinorbornene peptide. In addition, the cytocompatibility of the polymerization led to high postencapsulation viability of human mesenchymal stem cells, and the specificity of the tetrazine–norbornene reaction was exploited for sequential modification of the network via thiol–ene photochemistry. These advantages, combined with the synthetic accessibility of the tetrazine molecule compared to other bio-orthogonal click reagents, make this cross-linking chemistry an interesting and powerful new tool for the development of cell-instructive hydrogels for tissue engineering applications.

Photocontrolled nanoparticles for on-demand release of proteins.

We describe here light-regulated swelling and degradation features of polymeric nanoparticles that are produced using an inverse microemulsion polymerization method. We demonstrate the phototriggered release characteristics of the nanoparticles by sequestering protein molecules and releasing them using light as a trigger. Furthermore, the intracellular translocation of the nanoparticles, along with its fluorescent protein payload, was achieved using a cell-penetrating peptide-based surface modification. We expect that the noncovalent encapsulation of proteins using nanoparticles and their photo triggered release using an external light would provide opportunities for achieving intracellular release of molecular therapeutics for on-demand requirements.

Mechanically Stiff Nanocomposite Hydrogels at Ultralow Nanoparticle Content

Although hydrogels are able to mimic native tissue microenvironments, their utility for biomedical applications is severely hampered due to limited mechanical stiffness and low toughness. Despite recent progress in designing stiff and tough hydrogels, it is still challenging to achieve a cell-friendly, high modulus construct. Here, we report a highly efficient method to reinforce collagen-based hydrogels using extremely low concentrations of a nanoparticulate-reinforcing agent that acts as a cross-link epicenter. Extraordinarily, the addition of these nanoparticles at a 10 000-fold lower concentration relative to polymer resulted in a more than 10-fold increase in mechanical stiffness and a 20-fold increase in toughness. We attribute the high stiffness of the nanocomposite network to the chemical functionality of the nanoparticles, which enabled the cross-linking of multiple polymeric chains to the nanoparticle surface. The mechanical stiffness of the nanoengineered hydrogel can be tailored between 0.2 and 200 kPa simply by manipulating the size of the nanoparticles (4, 8, and 12 nm), as well as the concentrations of the nanoparticles and polymer. Moreover, cells can be easily encapsulated within the nanoparticulate-reinforced hydrogel network, showing high viability. In addition, encapsulated cells were able to sense and respond to matrix stiffness. Overall, these results demonstrate a facile approach to modulate the mechanical stiffness of collagen-based hydrogels and may have broad utility for various biomedical applications, including use as tissue-engineered scaffolds and cell/protein delivery vehicles.

A Diels-Alder modulated approach to control and sustain the release of dexamethasone and induce osteogenic differentiation of human mesenchymal stem cells.

We report a new approach to controlled drug release based upon exploiting the dynamic equilibrium that exists between Diels-Alder reactants and products, demonstrating the release of a furan containing dexamethasone peptide (dex-KGPQG-furan) from a maleimide containing hydrogel. Using a reaction-diffusion model, the release kinetics were tuned to achieve sustained concentrations conducive to osteogenic differentiation of human mesenchymal stem cells (hMSCs). Efficacy was first demonstrated in a 2D culture model, in which dexamethasone release induced significant increases in alkaline phosphatase (ALP) activity and mineral deposition in hMSCs compared to a dexamethasone-free treatment. The results were similar to that observed with a soluble dexamethasone treatment. More dramatic differences were observed in 3D culture, where co-encapsulation of a dexamethasone releasing hydrogel depot within an hMSC-laden extracellular matrix mimetic poly(ethylene glycol) hydrogel resulted in a local and robust osteogenic differentiation. ALP activity reached levels that were up to six times higher than the dexamethasone free treatment. Interestingly, at 5 and 10 day time points, the ALP activity exceeded the dexamethasone positive control, suggesting a potential benefit of sustained release in 3D culture. After 21 days, substantial mineralization comparable to the positive control was also observed in the hydrogels. Collectively, these results demonstrate Diels-Alder modulated release as an effective and versatile new platform for controlled drug delivery that may prove especially beneficial for sustaining the release of low molecular weight molecules in hydrogel systems.

Clickable, photodegradable hydrogels to dynamically modulate valvular interstitial cell phenotype.

Biophysical cues are widely recognized to influence cell phenotype. While this evidence was established using static substrates, there is growing interest in creating stimulus-responsive biomaterials that better recapitulate the dynamic extracellular matrix. Here, a clickable, photodegradable hydrogel substrate that allows the user to precisely control substrate elasticity and topography in situ is presented. The hydrogels are synthesized by reacting an 8-arm poly(ethylene glycol) alkyne with an azide-functionalized photodegradable crosslinker. The utility of this platform by exploiting its photoresponsive properties to modulate the phenotype of porcine aortic valvular interstitial cells (VICs) is demonstrated. First, VIC phenotype is monitored, in response to initial substratum modulus and static topographic cues. Higher modulus (E ≈ 15 kPa) substrates induce higher levels of activation (≈70% myofibroblasts) versus soft (E ≈ 3 kPa) substrates (≈20% myofibroblasts). Microtopographies that induce VIC alignment and elongation on low modulus substrates also stimulate activation. Finally, VIC phenotype is monitored in response to sequential in situ manipulations. The results illustrate that VIC activation on stiff surfaces (≈70% myofibroblasts) can be partially reversed by reducing surface modulus (≈30% myofibroblats) and subsequently re-activated by anisotropic topographies (≈60% myofibroblasts). Such dynamic substrates afford unique opportunities to decipher the complex role of matrix cues on the plasticity of VIC activation.

Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate.

Incorporation of degradable moieties into synthetic hydrogels has greatly increased the utility of these three-dimensional matrices for in vitro cell culture as well as tissue engineering applications. A common method for introducing degradability is the inclusion of oligopeptides sensitive to cleavage by matrix metalloproteinases (MMPs), enabling cell-mediated remodeling and migration within the material. While this strategy has been effective, characterization and measurement of cell-mediated degradation in these materials has remained challenging. There are 20+ MMP family members whose activity is regulated in space and time by a number of biochemical and biophysical cues. Thus, the typical approach of characterizing cleavage of degradable moieties in solution with recombinant enzymes does not easily translate to three-dimensional cell-mediated matrix remodeling. To address this challenge, we report here the synthesis of a cell-laden hydrogel matrix functionalized with a fluorogenic peptide substrate to provide real-time, quantitative monitoring of global MMP activity. Using this system, stimulation of MMP activity was observed with growth factor treatment in mammary epithelial cells and compared to classical zymography results. Further, the effect of biophysical cues on MMP activity of human mesenchymal stem cells was also investigated where more rigid hydrogels were observed to increase MMP activity. The regulation of MMP activity by these biochemical and biophysical cues highlights the need for in situ, real-time measurement of hydrogel degradation, and use of these functionalized hydrogels will aid in future rational design of degradable synthetic hydrogels for in vitro cell studies and tissue engineering applications.

Bio-inspired 3D microenvironments: a new dimension in tissue engineering

Biomaterial scaffolds have been a foundational element of the tissue engineering paradigm since the inception of the field. Over the years there has been a progressive move toward the rational design and fabrication of bio-inspired materials that mimic the composition as well as the architecture and 3D structure of tissues. In this review, we chronicle advances in the field that address key challenges in tissue engineering as well as some emerging applications. Specifically, a summary of the materials and chemistries used to engineer bio-inspired 3D matrices that mimic numerous aspects of the extracellular matrix is provided, along with an overview of bioprinting, an additive manufacturing approach, for the fabrication of engineered tissues with precisely controlled 3D structures and architectures. To emphasize the potential clinical impact of the bio-inspired paradigm in biomaterials engineering, some applications of bio-inspired matrices are discussed in the context of translational tissue engineering. However, focus is also given to recent advances in the use of engineered 3D cellular microenvironments for fundamental studies in cell biology, including photoresponsive systems that are shedding new light on how matrix properties influence cell phenotype and function. In an outlook for future work, the need for high-throughput methods both for screening and fabrication is highlighted. Finally, microscale organ-on-a-chip technologies are highlighted as a promising area for future investment in the application of bio-inspired microenvironments.

Bioactive hydrogels: Lighting the way.

Advances in photochemistry have profoundly impacted the way in which biology is studied. Now, a photoactivated enzymatic patterning method that offers spatiotemporal control over the presentation of bioactive proteins to direct cells in three-dimensional culture significantly expands the available chemical toolbox.

Facile and efficient Lewis acid catalyzed synthesis of an asymmetric tetrazine useful for bio-orthogonal click chemistry applications

Bio-orthogonal tetrazine click reactions have recently attracted significant interest for applications spanning biological imaging, cancer targeting, and biomaterials science. Here, we report a simple and efficient two-step scheme for the synthesis of an asymmetric tetrazine molecule containing a carboxylic acid handle for subsequent macromolecular conjugation. Yields as high as 75% were achieved using as little as 0.005 equivalents of nickel triflate catalyst, which is a significant improvement over previous methodologies.