Identification of GDF15 peptide fragments inhibiting GFRAL receptor signaling.

Growth differentiation factor 15 (GDF15) is believed to be a major causative factor for cancer-induced cachexia. Recent elucidation of the central circuits involved in GDF15 function and its signaling through the glial cell-derived neurotrophic factor family receptor α-like (GFRAL) has prompted the interest of targeting the GDF15-GFRAL signaling for energy homeostasis and body weight regulation. Here, we applied advanced peptide technologies to identify GDF15 peptide fragments inhibiting GFRAL signaling. SPOT peptide arrays revealed binding of GDF15 C-terminal peptide fragments to the extracellular domain of GFRAL. Parallel solid-phase peptide synthesis allowed for generation of complementary GDF15 peptide libraries and their subsequent functional evaluation in cells expressing the GFRAL/RET receptor complex. We identified a series of C-terminal fragments of GDF15 inhibiting GFRAL activity in the micromolar range. These novel GFRAL peptide inhibitors could serve as valuable tools for further development of peptide therapeutics towards the treatment of cachexia and other wasting disorders.

An Aldehyde Responsive, Cleavable Linker for Glucose Responsive Insulins.

A glucose responsive insulin (GRI) that responds to changes in blood glucose concentrations has remained an elusive goal. Here we describe the development of glucose cleavable linkers based on hydrazone and thiazolidine structures. We developed linkers with low levels of spontaneous hydrolysis but increased level of hydrolysis with rising concentrations of glucose, which demonstrated their glucose responsiveness in vitro. Lipidated hydrazones and thiazolidines were conjugated to the LysB29 side-chain of HI by pH-controlled acylations providing GRIs with glucose responsiveness confirmed in vitro for thiazolidines. Clamp studies showed increased glucose infusion at hyperglycemic conditions for one GRI indicative of a true glucose response. The glucose responsive cleavable linker in these GRIs allow changes in glucose levels to drive the release of active insulin from a circulating depot. We have demonstrated an unprecedented, chemically responsive linker concept for biopharmaceuticals.

Rapid Prototyping of Polymeric Nanopillars by 3D Direct Laser Writing for Controlling Cell Behavior.

Mammalian cells have been widely shown to respond to nano- and microtopography that mimics the extracellular matrix. Synthetic nano- and micron-sized structures are therefore of great interest in the field of tissue engineering, where polymers are particularly attractive due to excellent biocompatibility and versatile fabrication methods. Ordered arrays of polymeric pillars provide a controlled topographical environment to study and manipulate cells, but processing methods are typically either optimized for the nano- or microscale. Here, we demonstrate polymeric nanopillar (NP) fabrication using 3D direct laser writing (3D DLW), which offers a rapid prototyping across both size regimes. The NPs are interfaced with NIH3T3 cells and the effect of tuning geometrical parameters of the NP array is investigated. Cells are found to adhere on a wide range of geometries, but the interface depends on NP density and length. The Cell Interface with Nanostructure Arrays (CINA) model is successfully extended to predict the type of interface formed on different NP geometries, which is found to correlate with the efficiency of cell alignment along the NPs. The combination of the CINA model with the highly versatile 3D DLW fabrication thus holds the promise of improved design of polymeric NP arrays for controlling cell growth.

Mapping cell behavior across a wide range of vertical silicon nanocolumn densities.

Over the past decade, vertical nanostructures have provided novel approaches for biomedical applications such as intracellular delivery/detection, specific cell capture, membrane potential measurement, and cellular activity regulation. Although the feasibility of the vertical nanostructures as a new biological tool has been thoroughly demonstrated, a better understanding of cell behavior on vertical nanostructures, in particular the effects of geometry, is essential for advanced applications. To investigate the cell behavior according to the variation of the spacing between vertical nanostructures, we have interfaced fibroblasts (NIH3T3) with density-controlled vertical silicon nanocolumn arrays (vSNAs). Over a wide range of vSNA densities, we observe three distinct cell settling regimes and investigate both overall cell behavior (adhesions, morphology, and mobility) and detailed biomacromolecule variance (F-actin and focal adhesion) across these regimes. We expect that these systematic observations could serve as a guide for improved nanostructure array design for the desired cell manipulation.

Click Chemistry Mediated Functionalization of Vertical Nanowires for Biological Applications.

Semiconductor nanowires (NWs) are gaining significant importance in various biological applications, such as biosensing and drug delivery. Efficient and controlled immobilization of biomolecules on the NW surface is crucial for many of these applications. Here, we present for the first time the use of the Cu(I) -catalyzed alkyne-azide cycloaddition and its strain-promoted variant for the covalent functionalization of vertical NWs with peptides and proteins. The potential of the approach was demonstrated in two complementary applications of measuring enzyme activity and protein binding, which is of general interest for biological studies. The attachment of a peptide substrate provided NW arrays for the detection of protease activity. In addition, green fluorescent protein was immobilized in a site-specific manner and recognized by antibody binding to demonstrate the proof-of-concept for the use of covalently modified NWs for diagnostic purposes using minute amounts of material.

Covalent and stable CuAAC modification of silicon surfaces for control of cell adhesion.

Stable primary functionalization of metal surfaces plays a significant role in reliable secondary attachment of complex functional molecules used for the interfacing of metal objects and nanomaterials with biological systems. In principle, this can be achieved through chemical reactions either in the vapor or liquid phase. In this work, we compared these two methods for oxidized silicon surfaces and thoroughly characterized the functionalization steps by tagging and fluorescence imaging. We demonstrate that the vapor-phase functionalization only provided transient surface modification that was lost on extensive washing. For stable surface modification, a liquid-phase method was developed. In this method, silicon wafers were decorated with azides, either by silanization with (3-azidopropyl)triethoxysilane or by conversion of the amine groups of an aminopropylated surface by means of the azido-transfer reaction. Subsequently, D-amino acid adhesion peptides could be immobilized on the surface by use of Cu(I)-catalyzed click chemistry. This enabled the study of cell adhesion to the metal surface. In contrast to unmodified surfaces, the peptide-modified surfaces were able to maintain cell adhesion during significant flow velocities in a microflow reactor.

Exploring arrays of vertical one-dimensional nanostructures for cellular investigations.

The endeavor of exploiting arrays of vertical one-dimensional (1D) nanostructures (NSs) for cellular applications has recently been experiencing a pronounced surge of activity. The interest is rooted in the intrinsic properties of high-aspect-ratio NSs. With a height comparable to a mammalian cell, and a diameter 100-1000 times smaller, NSs should intuitively reach far into a cell and, due to their small diameter, do so without compromising cell health. Single NSs would thus be expedient for measuring and modifying cell response. Further organization of these structures into arrays can provide up-scaled and detailed spatiotemporal information on cell activity, an achievement that would entail a massive leap forward in disease understanding and drug discovery. Numerous proofs-of-principle published recently have expanded the large toolbox that is currently being established in this rapidly advancing field of research. Encouragingly, despite the diversity of NS platforms and experimental conditions used thus far, general trends and conclusions from combining cells with NSs are beginning to crystallize. This review covers the broad spectrum of NS materials and dimensions used; the observed cellular responses with specific focus on adhesion, morphology, viability, proliferation, and migration; compares the different approaches used in the field to provide NSs with the often crucial cytosolic access; covers the progress toward biological applications; and finally, envisions the future of this technology. By maintaining the impressive rate and quality of recent progress, it is conceivable that the use of vertical 1D NSs may soon be established as a superior choice over other current techniques, with all the further benefits that may entail.

Tuning InAs nanowire density for HEK293 cell viability, adhesion, and morphology: perspectives for nanowire-based biosensors.

Arrays of nanowires (NWs) are currently being established as vehicles for molecule delivery and electrical- and fluorescence-based platforms in the development of biosensors. It is conceivable that NW-based biosensors can be optimized through increased understanding of how the nanotopography influences the interfaced biological material. Using state-of-the-art homogenous NW arrays allow for a systematic investigation of how the broad range of NW densities used by the community influences cells. Here it is demonstrated that indium arsenide NW arrays provide a cell-promoting surface, which affects both cell division and focal adhesion up-regulation. Furthermore, a systematic variation in NW spacing affects both the detailed cell morphology and adhesion properties, where the latter can be predicted based on changes in free-energy states using the proposed theoretical model. As the NW density influences cellular parameters, such as cell size and adhesion tightness, it will be important to take NW density into consideration in the continued development of NW-based platforms for cellular applications, such as molecule delivery and electrical measurements.