Publications

Three-dimensional printing at the micro-/nanoscale represents a new challenge in research and development to achieve direct printing down to nanometre-sized objects. Here, FluidFM, a combination of microfluidics with atomic force microscopy, offers attractive options to fabricate hierarchical polymer structures at different scales. However, little is known about the effect of the substrate on the printed structures and the integration of (bio)functional groups into the polymer inks. In this study, we printed micro-/nanostructures on surfaces with different wetting properties, and integrated molecules with different functional groups (rhodamine as a fluorescent label and biotin as a binding tag for proteins) into the base polymer ink. The substrate wetting properties strongly affected the printing results, in that the lateral feature sizes increased with increasing substrate hydrophilicity. Overall, ink modification only caused minor changes in the stiffness of the printed structures. This shows the generality of the approach, as significant changes in the mechanical properties on chemical functionalization could be confounders in bioapplications. The retained functionality of the obtained structures after UV curing was demonstrated by selective binding of streptavidin to the printed structures. The ability to incorporate binding tags to achieve specific interactions between relevant proteins and the fabricated micro-/nanostructures, without compromising the mechanical properties, paves a way for numerous bio and sensing applications. Additional flexibility is obtained by tuning the substrate properties for feature size control, and the option to obtain functionalized printed structures without post-processing procedures will contribute to the development of 3D printing for biological applications, using FluidFM and similar dispensing techniques.

Supramolecular materials provide unique opportunities to mimic both the structure and mechanics of the biopolymer networks that compose the extracellular matrix. However, strategies to modify their filamentous structures in space and time in 3D cell culture to study cell behavior as encountered in development and disease are lacking. We herein disclose a multicomponent squaramide-based supramolecular material whose mechanics and bioactivity can be controlled by light through co-assembly of a 1,2-dithiolane (DT) monomer that forms disulfide cross-links. Remarkably, increases in storage modulus from ∼200 Pa to >10 kPa after stepwise photo-cross-linking can be realized without an initiator while retaining colorlessness and clarity. Moreover, viscoelasticity and plasticity of the supramolecular networks decrease upon photo-irradiation, reducing cellular protrusion formation and motility when performed at the onset of cell culture. When applied during 3D cell culture, force-mediated manipulation is impeded and cells move primarily along earlier formed channels in the materials. Additionally, we show photopatterning of peptide cues in 3D using either a photomask or direct laser writing. We demonstrate that these squaramide-based filamentous materials can be applied to the development of synthetic and biomimetic 3D in vitro cell and disease models, where their secondary cross-linking enables mechanical heterogeneity and shaping at multiple length scales.

Cancer cells circulating in blood vessels activate platelets, forming a cancer cell encircling platelet cloak which facilitates cancer metastasis. Heparin (H) is frequently used as an anticoagulant in cancer patients but up to 5% of patients have a side effect, heparin-induced thrombocytopenia (HIT) that can be life-threatening. HIT is developed due to a complex interaction among multiple components including heparin, platelet factor 4 (PF4), HIT antibodies, and platelets. However, available information regarding the effect of HIT components on cancers is limited. Here, we investigated the effect of these materials on the mechanical property of breast cancer cells using atomic force microscopy (AFM) while cell spreading was quantified by confocal laser scanning microscopy (CLSM), and cell proliferation rate was determined. Over time, we found a clear effect of each component on cell elasticity and cell spreading. In the absence of platelets, HIT antibodies inhibited cell proliferation but they promoted cell proliferation in the presence of platelets. Our results indicate that HIT complexes influenced the development of breast cancer cells.

Heparin-induced thrombocytopenia (HIT) antibodies (Abs) can mediate and activate blood cells, forming blood clots. To detect HIT Abs, immunological assays with high sensitivity (≥95%) and fast response are widely used, but only about 50% of these tests are accurate as non-HIT Abs also bind to the same antigens. We aim to develop biosensor-based electrical detection to better differentiate HIT-like from non-HIT-like Abs. As a proof of principle, we tested with two types of commercially available monoclonal Abs including KKO (inducing HIT) and RTO (noninducing HIT). Platelet factor 4/Heparin antigens were immobilized on gold electrodes, and binding of antibodies on the chips was detected based on the change in the charge transfer resistance (Rct). Binding of KKO on sensors yielded a significantly lower charge transfer resistance than that of RTO. Bound antibodies and their binding characteristics on the sensors were confirmed and characterized by complementary techniques. Analysis of thermal kinetics showed that RTO bonds are more stable than those of KKO, whereas KKO exhibited a higher negative ζ potential than RTO. These different characteristics made it possible to electrically differentiate these two types of antibodies. Our study opens a new avenue for the development of sensors for better detection of pathogenic Abs in HIT patients.

A major challenge in the use of HepG2 cell culture models for drug toxicity screening is their lack of maturity in 2D culture. 3D culture in Matrigel promotes the formation of spheroids that express liver-relevant markers, yet they still lack various primary hepatocyte functions. Therefore, alternative matrices where chemical composition and materials properties are controlled to steer maturation of HepG2 spheroids remain desired. Herein, a modular approach is taken based on a fully synthetic and minimalistic supramolecular matrix based on squaramide synthons outfitted with a cell-adhesive peptide, RGD for 3D HepG2 spheroid culture. Co-assemblies of RGD-functionalized squaramide-based and native monomers resulted in soft and self-recovering supramolecular hydrogels with a tunable RGD concentration. HepG2 spheroids are self-assembled and grown ( 150 m) within the supramolecular hydrogels with high cell viability and differentiation over 21 days of culture. Importantly, significantly higher mRNA and protein expression levels of phase I and II metabolic enzymes, drug transporters, and liver markers are found for the squaramide hydrogels in comparison to Matrigel. Overall, the fully synthetic squaramide hydrogels are proven to be synthetically accessible and effective for HepG2 differentiation showcasing the potential of this supramolecular matrix to rival and replace naturally-derived materials classically used in high-throughput toxicity screening.