Unravelling Receptor and RGD Motif Dependence of Retargeted Adenoviral Vectors using Advanced Tumor Model Systems.

Recent advances in engineering adenoviruses are paving the way for new therapeutic gene delivery approaches in cancer. However, there is limited knowledge regarding the impact of adenoviral retargeting on transduction efficiency in more complex tumor architectures, and the role of the RGD loop at the penton base in retargeting is unclear. To address this gap, we used tumor models of increasing complexity to study the role of the receptor and the RGD motif. Employing tumor-fibroblast co-culture models, we demonstrate the importance of the RGD motif for efficient transduction in 2D through the epithelial cell adhesion molecule (EpCAM), but not the epidermal growth factor receptor (EGFR). Via optical clearing of co-culture spheroids, we show that the RGD motif is required for transduction via both receptors in 3D tumor architectures. We subsequently employed a custom-designed microfluidic model containing collagen-embedded tumor spheroids, mimicking the interplay between interstitial flow, extracellular matrix and adenoviral transduction. Image analysis of on-chip cleared spheroids indicated the importance of the RGD motif for on-chip adenoviral transduction. Together, our results show the interrelationship between receptor characteristics, the RGD motif, the 3D tumor architecture and retargeted adenoviral transduction efficiency. The findings are important for the rational design of next-generation therapeutic adenoviruses.

DNA-functionalized hydrogels for confined membrane-free in vitro transcription/translation.

We microfluidically fabricate bio-orthogonal DNA-functionalized porous hydrogels from hyaluronic acid that are employed in in vitro transcription/translation (IVTT) of a green fluorescent protein. By co-encapsulating individual hydrogel particles and the IVTT machinery in water-in-oil microdroplets, we study protein expression in a defined reaction volume. Our approach enables precise control over protein expression rates by gene dosage. We show that gene transcription and translation are confined to the membrane-free hydrogel matrix, which contributes to the design of membrane-free protocells.

Artificial microniches for probing mesenchymal stem cell fate in 3D.

Droplet microfluidics is combined with bio-orthogonal thiol-ene click chemistry to fabricate micrometer-sized, monodisperse fibrinogen-containing hyaluronic acid hydrogel microbeads in a mild, radical-free procedure in the presence of human mesenchymal stem cells (hMSCs). The gel beads serve as microniches for the 3D culture of single hMSCs, containing hyaluronic acid and additional fibrinogen for cell surface binding, and they are porous and stable in tissue culture medium for up to 4 weeks with mechanical properties right in the range of soft solid tissues (0.9-9.2 kPa). The encapsulation procedure results in 70% viable hMSCs in the microbeads after 24 hours of culture and a very high degree of viability of the cells after long term culture of 2 weeks. hMSCs embedded in the microniches display an overall rounded morphology, consistent with those previously observed in 3D culture. Upon induction, the multipotency and differentiation potential of the hMSCs are characterized by staining of corresponding biomarkers, demonstrating a clear heterogeneity in the cell population. These hydrogel microbeads represent a versatile microstructured material platform with great potential for studying the differences of material cues and soluble factors in stem cell differentiation in a 3D tissue-like environment at the single cell level.

Dewetting of conducting polymer inkjet droplets on patterned surfaces.

The manufacture of high-performance electronic devices with micrometre or even submicrometre dimensions by solution processing and direct printing, requires the ability to control accurately the flow and spread of functional liquid inks on surfaces. This can be achieved with the help of surface-energy patterns causing inks to be repelled and dewetted from pre-defined regions of the substrate. To exploit this principle for the fabrication of submicrometre device structures, a detailed understanding of the factors causing ink droplets to dewet on patterned surfaces is required. Here, we use hydrophobic surface-energy barriers of different geometries to study the influence of solution viscosity, ink volume, and contact angle on the process of dewetting of inkjet-printed droplets of a water-based conducting polymer. We demonstrate polymer field-effect transistor devices with channel length of 500 nm fabricated by surface-energy-assisted inkjet printing.

Patterning electro-osmotic flow with patterned surface charge.

This Letter reports the measurement of electro-osmotic flows (EOF) in microchannels with surface charge patterned on the 200 microm scale. We have investigated two classes of patterns: (1) Those in which the surface charge varies along a direction perpendicular to the electric field used to drive the EOF; this type of pattern generates multidirectional flow along the direction of the field. (2) Those in which the surface charge pattern varies parallel to the field; this pattern generates recirculating cellular flow, and thus causes motion both parallel and perpendicular to the external field. Measurements of both of these flows agree well with theory in the limit of thin double layers and low surface potential.