Design and Fabrication of Organ-on-Chips: Promises and Challenges.

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.

Transferrin receptor targeting by de novo sheet extension.

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 Kd, 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.

Tumor-Derived Extracellular Vesicles Breach the Intact Blood-Brain Barrier via Transcytosis.

The restrictive nature of the blood-brain barrier (BBB) creates a major challenge for brain drug delivery with current nanomedicines lacking the ability to cross the BBB. Extracellular vesicles (EVs) have been shown to contribute to the progression of a variety of brain diseases including metastatic brain cancer and have been suggested as promising therapeutics and drug delivery vehicles. However, the ability of native tumor-derived EVs to breach the BBB and the mechanism(s) involved in this process remain unknown. Here, we demonstrate that tumor-derived EVs can breach the intact BBB in vivo, and by using state-of-the-art in vitro and in vivo models of the BBB, we have identified transcytosis as the mechanism underlying this process. Moreover, high spatiotemporal resolution microscopy demonstrated that the endothelial recycling endocytic pathway is involved in this transcellular transport. We further identify and characterize the mechanism by which tumor-derived EVs circumvent the low physiologic rate of transcytosis in the BBB by decreasing the brain endothelial expression of rab7 and increasing the efficiency of their transport. These findings identify previously unknown mechanisms by which tumor-derived EVs breach an intact BBB during the course of brain metastasis and can be leveraged to guide and inform the development of drug delivery approaches to deliver therapeutic cargoes across the BBB for treatment of a variety of brain diseases including, but not limited to, brain malignancies.

Optimizing design parameters of a peptide targeted liposomal nanoparticle in an in vivo multiple myeloma disease model after initial evaluation in vitro.

Despite ligand-targeted liposomes long garnering interest as drug delivery vehicles for cancer therapeutics, inconsistency in successful outcomes have hindered their translation into the clinic. This is in part due to discrepancies between in vitro design evaluations and final in vivo outcomes. By employing a multifaceted synthetic strategy to prepare peptide-targeted nanoparticles of high purity, reproducibility, and with precisely controlled quantity of functionalities, we systematically evaluated the individual roles that peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size play on cancer cell uptake and tumor targeting both in vitro and in vivo, and how the results correlated and contrasted. These parameters were analyzed using a VLA-4-targeted liposome system in a multiple myeloma mouse xenograft model to evaluate in vivo biodistribution and tumor cell uptake. The results showed that using in vitro models to optimize targeted-nanoparticles for maximum cellular uptake was helpful in narrowing down the particle characteristics. However, in vitro optimization fell short of achieving enhanced results in animal models, rather had negative consequences for in vivo targeting. This outcome is not surprising considering that the receptor being targeted is also present on healthy lymphocytes and increasing targeting peptide valency on particle surfaces results in an increase in non-selective, off-target binding to healthy cells. Hence, further optimization using in vivo models was absolutely necessary, through which we were able to increase the uptake of peptide-targeted liposomes by cancerous cells overexpressing VLA-4 to 15-fold over that of non-targeted liposomes in vivo. The results highlighted the importance of creating a comprehensive understanding of the effect of each liposome design parameter on multifactorial biological endpoints including both in vitro and in vivo in determining the therapeutic potential of peptide-targeted liposomes.

Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function and shuttling of drugs and antibodies.

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.

Site-specific conjugation of an antibody on a gold nanoparticle surface for one-step diagnosis of prostate specific antigen with dynamic light scattering.

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.

Antibody purification via affinity membrane chromatography method utilizing nucleotide binding site targeting with a small molecule.

Here, we present an affinity membrane chromatography technique for purification of monoclonal and polyclonal antibodies from cell culture media of hybridomas and ascites fluids. The m-NBST method utilizes the nucleotide-binding site (NBS) that is located on the Fab variable domain of immunoglobulins to enable capturing of antibody molecules on a membrane affinity column via a small molecule, tryptamine, which has a moderate binding affinity to the NBS. Regenerated cellulose membrane was selected as a matrix due to multiple advantages over traditionally used resin-based affinity systems. Rituximab was used for proof of concept experiments. Antibody purification was accomplished by first capture of injected samples while running equilibration buffer (50 mM sodium phosphate pH 7.0), followed by elution achieved by running a gradient of mild elution buffer (3 M NaCl in 50 mM phosphate pH 7.0). The results indicate that the m-NBST column efficiency for Rituximab was >98%, with a purity level of >98%. The quality and the capacity of this small molecule membrane affinity purification method is further evaluated for a number of parameters such as: injection concentrations, volumes, wash/bind time, elution gradient, antibody/protein-contaminant combinations, effects of injection buffer, post-purification antigen binding activity of antibodies, and column reusability and stability.

Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

Conjugation of a reactive thiol at the nucleotide binding site for site-specific antibody functionalization.

Described here is a UV photo-cross-linking method that utilizes the NBS (nucleotide binding site) for site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBS(Thiol)), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBS(Thiol) method by successfully functionalizing a prostate specific antigen antibody (IgG(PSA)) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5-1.5 J/cm(2) was determined to yield the most efficient photo-cross-linking and resulted in 1-1.5 conjugations per antibody while preserving antibody/antigen binding activity and Fc recognition. Utilizing the IBA-Thiol ligand allows for an efficient means of site-specifically conjugating UV sensitive functionalities to antibody NBS that would otherwise not have been amenable by the previously described UV-NBS photo-cross-linking method. The UV-NBS(Thiol) conjugation strategy can be utilized in various diagnostic and therapeutic applications with nearly limitless potential for the preparation of site-specific covalent conjugation of affinity tags, fluorescent molecules, peptides, and chemotherapeutics to antibodies.

Oriented antibody immobilization by site-specific UV photocrosslinking of biotin at the conserved nucleotide binding site for enhanced antigen detection.

The nucleotide binding site (NBS) is an under-utilized, highly conserved binding site found within the variable region of nearly all antibody Fab arms. Here, we describe an NBS specific UV photocrosslinking biotinylation method (UV-NBS(Biotin)) for the oriented immobilization of antibodies to streptavidin-coated surfaces, such that the antigen binding activity remains unaffected. An optimal UV exposure of 1J/cm(2) yielded an average conjugation efficiency of ≈ 1 biotin per antibody resulting in significant immobilization efficiency while maintaining maximal antigen binding activity. With the continued push for miniaturization of medical diagnostics to reduce cost and increase patient accessibility the ever shrinking on chip detection areas necessitate the highest level of immobilized antibody activity to maximize assay detection capabilities. The UV-NBS(Biotin) method yielded surfaces with significantly enhanced antigen detection capabilities, improved antigen detection sensitivity and the highest amount of active surface immobilized antibody when compared to other common immobilization methods including: ε-NH3(+) surface conjugation, NHS-Biotin, and direct physical adsorption. Taken together, the UV-NBS(Biotin) method provides a universal, site-specific immobilization method that is amenable to any available assay detection modality with potential significant implications in the development of miniaturized medical diagnostics and lab on a chip technologies.