Single-step purification of a small non-mAb biologic by peptide-ELP-based affinity precipitation.

Affinity precipitation using stimulus-responsive biopolymers such as elastin-like polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP-peptide fusion for the affinity precipitation of the therapeutically relevant small non-mAb biologic, AdP. A 12-mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and AdP interacted with a KD of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid-phase modifiers such as NaCl, arginine, or ethylene glycol was effective. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled >80% product recovery. The overall process performance evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase ultra-performance liquid chromatography analyses indicated successful single-step purification of the biologic from an Escherichia coli lysate resulting in ∼90% purity and >80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non-antibody biological product using ELP-based affinity precipitation.

Microarrays for the screening and identification of carbohydrate-binding peptides.

The development of carbohydrate-binding ligands is crucial for expanding knowledge on the glycocode and for achieving systematic carbohydrate targeting. Amongst such ligands, carbohydrate-binding peptides (CBPs) are attractive for use in bioanalytical and biomedical systems due to their biochemical and physicochemical properties; moreover, given the biological significance of lectin-carbohydrate interactions, these ligands offer an opportunity to study peptide sequence and binding characteristics to inform on natural target/ligand interactions. Here, a high-throughput microarray screening technique is described for the identification and study of CBPs, with a focus on polysialic acid (PSA), a polysaccharide found on neural stem cells. The chemical and biological uniqueness of PSA suggests that an ability to exclusively target this glycan may promote a number of diagnostic and therapeutic applications. PSA-binding peptides from phage display screening and from epitope mapping of an scFv for oligosialic acid were screened in an optimized microarray format with three ligand density conditions. Hypothesis-driven mutations were additionally applied to select peptides to modulate peptide affinity and selectivity to PSA. Peptide compositional and positional analyses revealed the significance of various residues for PSA binding and suggested the importance of basic residue positioning for PSA recognition. Furthermore, selectivity studies performed directly on microarrays with chondroitin sulfate A (CS-A) demonstrated the value of screening for both affinity and selectivity in the development of CBPs. Thus, the integrated approach described, with attention to design strategy, screening, and peptide characterization, successfully identified novel PSA-binding ligands and offers a platform for the identification and study of additional polysaccharide-binding peptides.

Combined Bioinformatic and Rational Design Approach To Develop Antimicrobial Peptides against Mycobacterium tuberculosis.

Drug-resistant pathogens are a growing problem, and novel strategies are needed to combat this threat. Among the most significant of these resistant pathogens is Mycobacterium tuberculosis, which is an unusually difficult microbial target due to its complex membrane. Here, we design peptides for specific activity against M. tuberculosis using a combination of "database filtering" bioinformatics, protein engineering, and de novo design. Several variants of these peptides are structurally characterized to validate the design process. The designed peptides exhibit potent activity (MIC values as low as 4 µM) against M. tuberculosis and also exhibit broad activity against a host of other clinically relevant pathogenic bacteria such as Gram-positive bacteria (Streptococcus) and Gram-negative bacteria (Escherichia coli). They also display excellent selectivity, with low cytotoxicity against cultured macrophages and lung epithelial cells. These first-generation antimicrobial peptides serve as a platform for the design of antibiotics and for investigating structure-activity relationships in the context of the M. tuberculosis membrane. The antimicrobial peptide design strategy is expected to be generalizable for any pathogen for which an activity database can be created.

Specificity of growth inhibitors and their cooperative effects in calcium oxalate monohydrate crystallization.

The molecular recognition and interactions governing site-specific adsorption of growth inhibitors on crystal surfaces can be tailored in order to control the anisotropic growth rates and physical properties of crystalline materials. Here we examine this phenomenon in calcium oxalate monohydrate (COM) crystallization, a model system of calcification with specific relevance for pathological mineralization. We analyzed the effect of three putative growth inhibitors--chondroitin sulfate, serum albumin, and transferrin--using analytical techniques capable of resolving inhibitor-crystal interactions from interfacial to bulk scales. We observed that each inhibitor alters surface growth by adsorbing on to distinct steps emanating from screw dislocations on COM surfaces. Binding of inhibitors to different crystallographic faces produced morphological modifications that are consistent with classical mechanisms of layer-by-layer crystal growth inhibition. The site-specific adsorption of inhibitors on COM surfaces was confirmed by bulk crystallization, fluorescent confocal microscopy, and atomic force microscopy. Kinetic studies of COM growth at varying inhibitor concentrations revealed marked differences in their efficacy and potency. Systematic analysis of inhibitor combinations, quantified via the combination index, identified various binary pairings capable of producing synergistic, additive, and antagonistic effects. Collectively, our investigation of physiologically relevant biomolecules suggests potential roles of COM inhibitors in pathological crystallization and provides guiding principles for biomimetic design of molecular modifiers for applications in crystal engineering.

Natural promoters of calcium oxalate monohydrate crystallization.

Crystallization is often facilitated by modifiers that interact with specific crystal surfaces and mediate the anisotropic rate of growth. Natural and synthetic modifiers tend to function as growth inhibitors that hinder solute attachment and impede the advancement of layers on crystal surfaces. There are fewer examples of modifiers that operate as growth promoters, whereby modifier-crystal interactions accelerate the kinetic rate of crystallization. Here, we examine two proteins, lysozyme and lactoferrin, which are observed in the organic matrix of three types of pathological stones: renal, prostatic, and pancreatic stones. This work focuses on the role of these proteins in the crystallization of calcium oxalate monohydrate (COM), the most prominent constituent of human kidney stones. Using a combination of experimental techniques, we show that these proteins, which are rich in l-arginine and l-lysine amino acids, promote COM growth. The synthesis and testing of peptides derived from contiguous segments of lysozyme's primary amino acid sequence revealed subdomains within the protein that operate either as an inhibitor or promoter of COM growth, with the latter exhibiting efficacies that nearly match that of the protein. We observed that cationic proteins promote COM growth over a wide range of modifier concentration, which differs from calcification promoters in the literature that exhibit dual roles as promoters and inhibitors at low and high concentration, respectively. This seems to suggest a unique mechanism of action for lysozyme and lactoferrin. Possible explanations for their effects on COM growth and crystal habit are proposed on the basis of classical colloidal theories and the physicochemical properties of peptide subdomains, including the number and spatial location of charged or hydrogen-bonding moieties.

Design of affinity peptides from natural protein ligands: A study of the cardiac troponin complex.

We describe a general strategy for the design and discovery of affinity peptides for a protein from its natural ligands. Our approach is guided by protein-protein interactions in natural systems and focuses on the hetero-trimeric complex of cardiac troponin I (cTnI), C (cTnC) and T (cTnT). A key premise of this work is that cTnC and cTnT, owing to their innate ability to bind cTnI, are potential templates for the design and discovery of cTnI-binding peptides. Relying only on the knowledge of primary sequences of cTnC and cTnT, we designed a library of short overlapping peptides that span the entirety of cTnC and cTnT and tested them for binding to cTnI. We were successful in identifying several peptides that display high affinity (1-100 nM) for cTnI. The specific implication of this work is that mimicking natural protein-protein interactions is an excellent starting point for the discovery and rational design of peptide ligands. The knowledge of secondary or tertiary structures of the proteins involved is not a necessary precondition for this approach. Nevertheless, we show that structural information can be used to validate the results of a fragment-based peptide design, and can be potentially beneficial for refining the lead candidates. Our approach is broadly applicable to any protein with at least one natural binding ligand with known primary sequence. For protein targets with multiple natural ligands, this approach can potentially yield several distinct affinity peptides capable of simultaneously binding the target protein via orthogonal modes or at complementary interfaces.

A Novel 3D Printed Model of Infected Human Hair Follicles to Demonstrate Targeted Delivery of Nanoantibiotics.

Hair follicle-penetrating nanoparticles offer a promising avenue for targeted antibiotic delivery, especially in challenging infections like acne inversa or folliculitis decalvans. However, demonstrating their efficacy with existing preclinical models remains difficult. This study presents an innovative approach using a 3D in vitro organ culture system with human hair follicles to investigate the hypothesis that antibiotic nanocarriers may reach bacteria within the follicular cleft more effectively than free drugs. Living human hair follicles were transplanted into a collagen matrix within a 3D printed polymer scaffold to replicate the follicle's microenvironment. Hair growth kinetics over 7 days resembled those of simple floating cultures. In the 3D model, fluorescent nanoparticles exhibited some penetration into the follicle, not observed in floating cultures. Staphylococcus aureus bacteria displayed similar distribution profiles postinfection of follicles. While rifampicin-loaded lipid nanocapsules were as effective as free rifampicin in floating cultures, only nanoencapsulated rifampicin achieved the same reduction of CFU/mL in the 3D model. This underscores the hair follicle microenvironment's critical role in limiting conventional antibiotic treatment efficacy. By mimicking this microenvironment, the 3D model demonstrates the advantage of topically administered nanocarriers for targeted antibiotic therapy against follicular infections.

Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Design of peptide affinity ligands for S-protein: a comparison of combinatorial and de novo design strategies.

Design of peptide affinity ligands against biological targets is important for a broad range of applications. Here, we report on de novo and combinatorial strategies for the design of high-affinity and high-specificity peptides against S-protein as a target. The peptide libraries employed in this study contain (1) consensus motif (CM) sequences identified from high-throughput phage combinatorial screening, (2) point mutations of CM sequences, and (3) de novo sequences rationally designed based on stereo-chemical information of the complex between S-protein and its natural ligand, S-peptide. In general, point mutations to CM allowed for modulating peptide affinity and specificity over a broad range. This is particularly useful in designing peptides with varying affinities and specificities for the target. De novo sequences, especially those based on the S-protein binding pocket, on average bound with higher affinities within a narrow range (10-100 nM) as compared to point mutations to CM (1 nM-2 µM). As such, the approaches described here serve as a general guide for optimizing the design of peptide affinity ligands for a wide range of target proteins or applications.

Incorporation of hair follicles in 3D bioprinted models of human skin.

Current approaches fail to adequately introduce complex adnexal structures such as hair follicles within tissue engineered models of skin. Here, we report on the use of 3D bioprinting to incorporate these structures in engineered skin tissues. Spheroids, induced by printing dermal papilla cells (DPCs) and human umbilical vein cells (HUVECs), were precisely printed within a pregelled dermal layer containing fibroblasts. The resulting tissue developed hair follicle-like structures upon maturation, supported by migration of keratinocytes and melanocytes, and their morphology and composition grossly mimicked that of the native skin tissue. Reconstructed skin models with increased complexity that better mimic native adnexal structures can have a substantial impact on regenerative medicine as grafts and efficacy models to test the safety of chemical compounds.

Manufacturing of Pure Iron by Cold Rolling and Investigation for Application in Magnetic Flux Shielding.

The presented work investigates a novel method to manufacture 98.8% pure iron strips having high permeability and better saturation flux density for application in magnetic flux shielding. The proposed method uses electro-deposition and cold rolling along with intermediate annealing in a controlled environment to manufacture 0.05-0.5 mm thick pure iron strips. The presented approach is inexpensive, has better control over scaling/oxidation and requires low energy than that of the conventional methods of pure iron manufacturing by pyrometallurgical methods. Important magnetic and mechanical properties of the pure iron are investigated in the context of the application of the material in magnetic shielding. Magnetic properties of the material are investigated by following IEC60404-4 standard and toroidal coil test to determine hysteresis curve, magnetic permeability and core losses. The microstructure is investigated with an optical microscope and scanning electron microscopy to study grain size and defects after cold rolling and annealing. The properties derived from the experimental methods are used in finite element analysis to study the application of the material for static, low-frequency and high-frequency magnetic shielding. Theoretical simulation results for magnetic shielding around a current-carrying conductor and micro-electromechanical inductive sensor system are discussed. Further shielding performance of the material is compared with that of the other candidate materials, including that of Mu-metal and electrical steel. It is demonstrated that the pure iron strips manufactured in the present study can be used for magnetic shielding in the case of low-frequency applications. In the case of high-frequency applications, a conducting layer can be combined to ensure the required shielding effectiveness in the case of Class 2 applications.

Bioengineered Efficacy Models of Skin Disease: Advances in the Last 10 Years.

Models of skin diseases, such as psoriasis and scleroderma, must accurately recapitulate the complex microenvironment of human skin to provide an efficacious platform for investigation of skin diseases. Skin disease research has been shifting from less complex and less relevant 2D (two-dimensional) models to significantly more relevant 3D (three-dimensional) models. Three-dimensional modeling systems are better able to recapitulate the complex cell-cell and cell-matrix interactions that occur in vivo within skin. Three-dimensional human skin equivalents (HSEs) have emerged as an advantageous tool for the study of skin disease in vitro. These 3D HSEs can be highly complex, containing both epidermal and dermal compartments with integrated adnexal structures. The addition of adnexal structures to 3D HSEs has allowed researchers to gain more insight into the complex pathology of various hereditary and acquired skin diseases. One method of constructing 3D HSEs, 3D bioprinting, has emerged as a versatile and useful tool for generating highly complex HSEs. The development of commercially available 3D bioprinters has allowed researchers to create highly reproducible 3D HSEs with precise integration of multiple adnexal structures. While the field of bioengineered models for study of skin disease has made tremendous progress in the last decade, there are still significant efforts necessary to create truly biomimetic skin disease models. In future studies utilizing 3D HSEs, emphasis must be placed on integrating all adnexal structures relevant to the skin disease under investigation. Thorough investigation of the intricate pathology of skin diseases and the development of effective treatments requires use of highly efficacious models of skin diseases.

Evaluation of native and non-native biomaterials for engineering human skin tissue.

A variety of human skin models have been developed for applications in regenerative medicine and efficacy studies. Typically, these employ matrix molecules that are derived from non-human sources along with human cells. Key limitations of such models include a lack of cellular and tissue microenvironment that is representative of human physiology for efficacy studies, as well as the potential for adverse immune responses to animal products for regenerative medicine applications. The use of recombinant extracellular matrix proteins to fabricate tissues can overcome these limitations. We evaluated animal- and non-animal-derived scaffold proteins and glycosaminoglycans for the design of biomaterials for skin reconstruction in vitro. Screening of proteins from the dermal-epidermal junction (collagen IV, laminin 5, and fibronectin) demonstrated that certain protein combinations when used as substrates increase the proliferation and migration of keratinocytes compared to the control (no protein). In the investigation of the effect of components from the dermal layer (collagen types I and III, elastin, hyaluronic acid, and dermatan sulfate), the primary influence on the viability of fibroblasts was attributed to the source of type I collagen (rat tail, human, or bovine) used as scaffold. Furthermore, incorporation of dermatan sulfate in the dermal layer led to a reduction in the contraction of tissues compared to the control where the dermal scaffold was composed primarily of collagen type I. This work highlights the influence of the composition of biomaterials on the development of complex reconstructed skin models that are suitable for clinical translation and in vitro safety assessment.

Design of optimized diffusion-controlled transdermal drug delivery systems.

BACKGROUND: We describe a systematic approach to designing multilayer transdermal patches based on therapeutically relevant specifications of the drug. METHOD: Random search optimization techniques are used to optimize maximum drug release from the patch subject to the therapeutic specifications. Barrier layer thickness and relative concentrations of the drug in the drug-containing layers are used as key design parameters. RESULTS: A patch made of two drug-containing layers of equal thicknesses and relative drug concentrations of 20% and 80%, and a barrier layer with thickness of 14% compared to the total thickness of drug-containing layers was found to be the most optimum design. CONCLUSION: The proposed design is almost universally applicable and satisfies therapeutically relevant specifications while maximizing drug utilization.

Enhancement of transdermal drug delivery via synergistic action of chemicals.

Transdermal drug delivery is an attractive alternative to conventional techniques for administration of systemic therapeutics. One challenge in designing transdermal drug delivery systems is to overcome the natural transport barrier of the skin. Chemicals offer tremendous potential in overcoming the skin barrier to enhance transport of drug molecules. Individual chemicals are however limited in their efficacy in disrupting the skin barrier at low concentrations and usually cause skin irritation at high concentrations. Multicomponent mixtures of chemicals, however, have been shown to provide high skin permeabilization potency as compared to individual chemicals without necessarily causing irritation. Here we review systems employing synergistic mixtures of chemicals that offer superior skin permeation enhancement. These synergistic systems include solvent mixtures, microemulsions, eutectic mixtures, complex self-assembled vesicles and inclusion complexes. Methods for design and discovery of such synergistic systems are also discussed.

Purification of proteins using peptide-ELP based affinity precipitation.

In this work, we employed fusions of affinity peptides and elastin-like polypeptide (ELP) to carry out a proof-of-concept study for the single-step purification of model, tag-free proteins. Three known peptide-protein binding pairs of varied binding strengths were evaluated. The peptide-protein binding was first characterized through in-solution binding techniques such as fluorescence spectroscopy. Peptide-ELP constructs were then produced in E. coli and purified by phase transition. The binding of the peptide-ELP constructs to the products were then evaluated using competitive fluorescence spectroscopy. Affinity capture, precipitation and recovery experiments were then conducted using the three constructs to evaluate the efficacy of these peptide-ELP based affinity precipitation processes. Two out of the three systems tested were successful in capturing the product and yielding pure protein in a single affinity precipitation step. These results indicated that peptide-protein affinity played an important role in both the effective capture of the protein, and also in the required binding molar ratio for the affinity precipitation process. In addition, for intermediate affinity systems, constructs containing two copies of the peptide at the N-terminus of the ELP were beneficial towards achieving both high purity and yield, likely due to increased affinity from avidity effects. This proof-of-concept study lays the foundations for the development of new peptide-ELP-based affinity purification processes for industrially relevant proteins and other classes of biologics.

Rational identification and characterisation of peptide ligands for targeting polysialic acid.

The alpha-2,8-linked form of the polysaccharide polysialic acid (PSA) has widespread implications in physiological and pathological processes, ranging from neurological development to disease progression. Though the high electronegativity and excluded volume of PSA often promotes interference of biomolecular interactions, PSA-binding ligands have important implications for both biological processes and biotechnological applications. As such, the design, identification, and characterisation of novel ligands towards PSA is critical for expanding knowledge of PSA interactions and achieving selective glycan targeting. Here, we report on a rational approach for the identification of alpha-2,8-PSA-binding peptides, involving design from the endogenous ligand Siglec-11 and multi-platform characterisation of peptide binding. Microarray-based examination of peptides revealed charge and sequence characteristics influencing peptide affinity to PSA, and carbohydrate-peptide binding was further quantified with a novel fluorescence anisotropy assay. PSA-binding peptides exhibited specific binding to polymeric SA, as well as different degrees of selective binding in various conditions, including competition with PSA of alternating 2,8/9-linkages and screening with PSA-expressing cells. A computational study of Siglec-11 and Siglec-11-derived peptides offered synergistic insight into ligand binding. These results demonstrate the potential of PSA-binding peptides for selective targeting and highlight the importance of the approaches described herein for the study of carbohydrate interactions.

Three Dimensional Bioprinting of a Vascularized and Perfusable Skin Graft Using Human Keratinocytes, Fibroblasts, Pericytes, and Endothelial Cells.

Multilayered skin substitutes comprising allogeneic cells have been tested for the treatment of nonhealing cutaneous ulcers. However, such nonnative skin grafts fail to permanently engraft because they lack dermal vascular networks important for integration with the host tissue. In this study, we describe the fabrication of an implantable multilayered vascularized bioengineered skin graft using 3D bioprinting. The graft is formed using one bioink containing human foreskin dermal fibroblasts (FBs), human endothelial cells (ECs) derived from cord blood human endothelial colony-forming cells (HECFCs), and human placental pericytes (PCs) suspended in rat tail type I collagen to form a dermis followed by printing with a second bioink containing human foreskin keratinocytes (KCs) to form an epidermis. In vitro, KCs replicate and mature to form a multilayered barrier, while the ECs and PCs self-assemble into interconnected microvascular networks. The PCs in the dermal bioink associate with EC-lined vascular structures and appear to improve KC maturation. When these 3D printed grafts are implanted on the dorsum of immunodeficient mice, the human EC-lined structures inosculate with mouse microvessels arising from the wound bed and become perfused within 4 weeks after implantation. The presence of PCs in the printed dermis enhances the invasion of the graft by host microvessels and the formation of an epidermal rete. Impact Statement Three Dimensional printing can be used to generate multilayered vascularized human skin grafts that can potentially overcome the limitations of graft survival observed in current avascular skin substitutes. Inclusion of human pericytes in the dermal bioink appears to improve both dermal and epidermal maturation.

Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments.

In this work, two phage biopanning strategies were developed to identify affinity peptides for a single Fab and multiple kappa Fabs. For the biopanning rounds, protein L beads were employed to bind Fab targets in a fixed orientation, and NHS functionalized magnetic beads were used to facilitate evaluation of low pH elution conditions. The resulting peptide sequences were synthesized and the binding to different Fabs was evaluated using fluorescence polarization. The first biopanning approach yielded a peptide with similar affinities for two forms of the Fab (recombinantly expressed and post papain-digestion) as well as the intact antibody. While moderate affinity was observed toward a murine variant of the Fab with the same complementarity determining regions (CDR) region but different framework, minimal binding occurred to a Fab with high sequence homology but containing different CDR loops. The second biopanning strategy yielded a peptide with affinity for all three kappa Fabs indicating that it may be a good lead for the development of more general affinity reagents for recombinant kappa Fabs. Finally, an affinity peptide column was developed, and its efficacy was demonstrated for Fab purification from a complex cell culture fluid mixture. The results presented in this article demonstrate that different peptide-based phage biopanning strategies can be effectively employed to identify affinity peptide leads for specific Fab and more general kappa Fab purifications.

A microfluidic model of human brain (µHuB) for assessment of blood brain barrier.

Microfluidic cellular models, commonly referred to as "organs-on-chips," continue to advance the field of bioengineering via the development of accurate and higher throughput models, captivating the essence of living human organs. This class of models can mimic key in vivo features, including shear stresses and cellular architectures, in ways that cannot be realized by traditional two-dimensional in vitro models. Despite such progress, current organ-on-a-chip models are often overly complex, require highly specialized setups and equipment, and lack the ability to easily ascertain temporal and spatial differences in the transport kinetics of compounds translocating across cellular barriers. To address this challenge, we report the development of a three-dimensional human blood brain barrier (BBB) microfluidic model (µHuB) using human cerebral microvascular endothelial cells (hCMEC/D3) and primary human astrocytes within a commercially available microfluidic platform. Within µHuB, hCMEC/D3 monolayers withstood physiologically relevant shear stresses (2.73 dyn/cm2) over a period of 24 hr and formed a complete inner lumen, resembling in vivo blood capillaries. Monolayers within µHuB expressed phenotypical tight junction markers (Claudin-5 and ZO-1), which increased expression after the presence of hemodynamic-like shear stress. Negligible cell injury was observed when the monolayers were cultured statically, conditioned to shear stress, and subjected to nonfluorescent dextran (70 kDa) transport studies. µHuB experienced size-selective permeability of 10 and 70 kDa dextrans similar to other BBB models. However, with the ability to probe temporal and spatial evolution of solute distribution, µHuBs possess the ability to capture the true variability in permeability across a cellular monolayer over time and allow for evaluation of the full breadth of permeabilities that would otherwise be lost using traditional end-point sampling techniques. Overall, the µHuB platform provides a simplified, easy-to-use model to further investigate the complexities of the human BBB in real-time and can be readily adapted to incorporate additional cell types of the neurovascular unit and beyond.