Nathan A. Hotaling

Biomaterial Strategies for Immunomodulation

Strategies to enhance, suppress, or qualitatively shape the immune response are of importance for diverse biomedical applications, such as the development of new vaccines, treatments for autoimmune diseases and allergies, strategies for regenerative medicine, and immunotherapies for cancer. However, the intricate cellular and molecular signals regulating the immune system are major hurdles to predictably manipulating the immune response and developing safe and effective therapies. To meet this challenge, biomaterials are being developed that control how, where, and when immune cells are stimulated in vivo, and that can finely control their differentiation in vitro. We review recent advances in the field of biomaterials for immunomodulation, focusing particularly on designing biomaterials to provide controlled immunostimulation, targeting drugs and vaccines to lymphoid organs, and serving as scaffolds to organize immune cells and emulate lymphoid tissues. These ongoing efforts highlight the many ways in which biomaterials can be brought to bear to engineer the immune system.

Microfluidic Thrombosis under Multiple Shear Rates and Antiplatelet Therapy Doses

The mainstay of treatment for thrombosis, the formation of occlusive platelet aggregates that often lead to heart attack and stroke, is antiplatelet therapy. Antiplatelet therapy dosing and resistance are poorly understood, leading to potential incorrect and ineffective dosing. Shear rate is also suspected to play a major role in thrombosis, but instrumentation to measure its influence has been limited by flow conditions, agonist use, and non-systematic and/or non-quantitative studies. In this work we measured occlusion times and thrombus detachment for a range of initial shear rates (500, 1500, 4000, and 10000 s−1) and therapy concentrations (0–2.4 µM for eptifibatide, 0–2 mM for acetyl-salicylic acid (ASA), 3.5–40 Units/L for heparin) using a microfluidic device. We also measured complete blood counts (CBC) and platelet activity using whole blood impedance aggregometry. Effects of shear rate and dose were analyzed using general linear models, logistic regressions, and Cox proportional hazards models. Shear rates have significant effects on thrombosis/dose-response curves for all tested therapies. ASA has little effect on high shear occlusion times, even at very high doses (up to 20 times the recommended dose). Under ASA therapy, thrombi formed at high shear rates were 4 times more prone to detachment compared to those formed under control conditions. Eptifibatide reduced occlusion when controlling for shear rate and its efficacy increased with dose concentration. In contrast, the hazard of occlusion from ASA was several orders of magnitude higher than that of eptifibatide. Our results show similar dose efficacy to our low shear measurements using whole blood aggregometry. This quantitative and statistically validated study of the effects of a wide range of shear rate and antiplatelet therapy doses on occlusive thrombosis contributes to more accurate understanding of thrombosis and to models for optimizing patient treatment.

Molecular factors in dendritic cell responses to adsorbed glycoconjugates.

Carbohydrates and glycoconjugates have been shown to exert pro-inflammatory effects on the dendritic cells (DCs), supporting pathogen-induced innate immunity and antigen processing, as well as immunosuppressive effects in the tolerance to self-proteins. Additionally, the innate inflammatory response to implanted biomaterials has been hypothesized to be mediated by inflammatory cells interacting with adsorbed proteins, many of which are glycosylated. However, the molecular factors relevant for surface displayed glycoconjugate modulation of dendritic cell (DC) phenotype are unknown. Thus, in this study, a model system was developed to establish the role of glycan composition, density, and carrier cationization state on DC response. Thiol modified glycans were covalently bound to a model protein carrier, maleimide functionalized bovine serum albumin (BSA), and the number of glycans per BSA modulated. Additionally, the carrier isoelectric point was scaled from a pI of ∼4.0 to ∼10.0 using ethylenediamine (EDA). The DC response to the neoglycoconjugates adsorbed to wells of a 384-well plate was determined via a high throughput assay. The underlying trends in DC phenotype in relation to conjugate properties were elucidated via multivariate general linear models. It was found that glycoconjugates with more than 20 glycans per carrier had the greatest impact on the pro-inflammatory response from DCs, followed by conjugates having an isoelectric point above 9.5. Surfaces displaying terminal α1-2 linked mannose structures were able to increase the inflammatory DC response to a greater extent than did any other terminal glycan structure. The results herein can be applied to inform the design of the next generation of combination products and biomaterials for use in future vaccines and implanted materials.

Presentation modality of glycoconjugates modulates dendritic cell phenotype

The comparative dendritic cell (DC) response to glycoconjugates presented in soluble, phagocytosable, or non-phagocytosable display modalities is poorly understood. This is particularly problematic, as the probing of immobilized glycans presented on the surface of microarrays is a common screen for potential candidates for glycan-based therapeutics. However, the assumption that carbohydrate-protein interactions on a flat surface can be translatable to development of efficacious therapies, such as vaccines, which are delivered in soluble or phagocytosable particles, has not been validated. Thus, a preliminary investigation was performed in which mannose or glucose was conjugated to cationized bovine serum albumin and presented to DCs in soluble, phagocytosable, or non-phagocytosable display modalities. The functional DC response to the glycoconjugates was assessed via a high throughput assay. Dendritic cell phenotypic outcomes were placed into a multivariate, general linear model (GLM) and shown to be statistically different amongst display modalities when comparing similar surface areas. The GLM showed that glycoconjugates that were adsorbed to wells were the most pro-inflammatory while soluble conjugates were the least. DC interactions with mannose conjugates were found to be calcium dependent and could be inhibited via anti-DC-SIGN antibodies. The results of this study aim to resolve conflicts in reports from multiple laboratories showing differential DC profiles in response to similar, if not identical, ligands delivered via different modalities. Additionally, this study begins to bridge the gap between microarray binding data and functional cell responses by highlighting the phenotypes induced from adsorbed glycoconjugates as compared to those in solution or displayed on microparticles.

A switchable positive and negative air pressure device for efficient and gentle handling of nanofiber scaffolds

A scaffold handling device (SHD) has been designed that can switch from gentle suction to positive pressure to lift and place nanofiber scaffolds. In tissue engineering laboratories, delicate fibrous scaffolds, such as electrospun nanofiber scaffolds, are often used as substrates for cell culture. Typical scaffold handling procedures include lifting the scaffolds, moving them from one container to another, sterilization, and loading scaffolds into cell culture plates. Using tweezers to handle the scaffolds can be slow, can damage the scaffolds, and can cause them to wrinkle or fold. Scaffolds may also acquire a static charge which makes them difficult to put down as they cling to tweezers. An SHD has been designed that enables more efficient, gentle lifting, and placement of delicate scaffolds. Most of the parts to make the SHD can be purchased, except for the tip which can be 3D-printed. The SHD enables more reliable handling of nanofiber scaffolds that may improve the consistency of biomanufacturing processes.