The high selectivity of the human blood-brain barrier (BBB) restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip BBB model lined by induced pluripotent stem cell-derived human brain microvascular endothelium interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins and functional efflux pumps, and it displays selective transcytosis of peptides and antibodies previously observed in vivo. Increased barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB.

The advent of the miniaturization approach has influenced the research trends in almost all disciplines. Bioengineering is one of the fields benefiting from the new possibilities of microfabrication techniques, especially in cell and tissue culture, disease modeling, and drug discovery. The limitations of existing 2D cell culture techniques, the high time and cost requirements, and the considerable failure rates have led to the idea of 3D cell culture environments capable of providing physiologically relevant tissue functions in vitro. Organ-on-chips are microfluidic devices used in this context as a potential alternative to in vivo animal testing to reduce the cost and time required for drug evaluation. This emerging technology contributes significantly to the development of various research areas, including, but not limited to, tissue engineering and drug discovery. However, it also brings many challenges. Further development of the technology requires interdisciplinary studies as some problems are associated with the materials and their manufacturing techniques. Therefore, in this paper, organ-on-chip technologies are presented, focusing on the design and fabrication requirements. Then, state-of-the-art materials and microfabrication techniques are described in detail to show their advantages and also their limitations. A comparison and identification of gaps for current use and further studies are therefore the subject of the final discussion.

Small dimensions of gold nanoparticles (AuNPs) necessitate antibodies to be immobilized in an oriented fashion in order to conserve their antigen binding activity for proper function. In this study, we used the previously described UV-NBS method to site-specifically incorporate a thioctic acid (TA) functionality into antibodies at the conserved nucleotide-binding site (NBS). Modified antibodies were immobilized on the AuNP surface in an oriented manner utilizing the newly incorporated TA functionality while maintaining the antibody structure and activity. The resulting antibody functionalized AuNPs via the UV-NBS method demonstrated significantly enhanced antigen detection capabilities and improved antigen detection sensitivity with a high level of selectivity when compared to other commonly used AuNP functionalization methods. Our results demonstrate that the limit of detection (LOD) for AuNPs functionalized via the UV-NBS method was 55 pM PSA, which is 40, 851, and 5873-fold improved over the other immobilization methods: EDC-NHS, thiol reduction, and ionic interaction, respectively. Consequently, the UV-NBS method provides a universal, site-specific functionalization method that generates highly sensitive and more stable antibody functionalized AuNPs which are amenable to any available detection and treatment assay with potential significant implications.

The de novo design of polar protein–protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood–brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM K_d, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands. Significance The de novo design of proteins that bind natural target proteins is useful for a variety of biomedical and biotechnological applications. We describe a design strategy to target proteins containing an exposed beta edge strand. We use the approach to design binders to the human transferrin receptor which shuttles back and forth across the blood–brain barrier. Such binders could be useful for the delivery of therapeutics into the brain.