Design and characterization of lipid nanocarriers for oral delivery of immunotherapeutic peptides.

The use of therapeutic proteins and peptides is of great interest for the treatment of many diseases, and advances in nanotechnology offer a path toward their stable delivery via preferred routes of administration. In this study, we sought to design and formulate a nanostructured lipid carrier (NLC) containing a nominal antigen (insulin peptide) for oral delivery. We utilized the design of experiments (DOE) statistical method to determine the dependencies of formulation variables on physicochemical particle characteristics including particle size, polydispersity (PDI), melting point, and latent heat of melting. The particles were determined to be non-toxic in vitro, readily taken up by primary immune cells, and found to accumulate in regional lymph nodes following oral administration. We believe that this platform technology could be broadly useful for the treatment of autoimmune diseases by supporting the development of oral delivery-based antigen specific immunotherapies.

Design of experiments approach to developing a robust ink for bioprinting.

Despite advancements in tissue engineering, the methods used to generate three-dimensional (3D)in vitromodels for rapid screening and characterization studies remain time and labor intensive. Bioprinting offers an opportunity to offset these limitations by providing a scalable, high-throughput method with precise control over biomaterial scaffold and cellular deposition. However, the process of formulating bioinks can be complex in terms of balancing the mechanical integrity of a bioscaffold and viability of cells. One key factor, especially in alginate-based bioinks, is the rate of bioscaffold dissolution. It must allow cells to replace the bioscaffold with extracellular matrix (ECM), yet remain durable during extended tissue culture. This study uses a Design of Experiments (DoE) approach to understand the dependencies of multiple variables involved in the formulation and processing of an alginate-based bioink. The focus of the DoE was to understand the effects of hydrogel composition on bioink durability while maintaining cell viability. Three ingredients were varied in all: alginate, nanocellulose, and fibrinogen. Their effects on the bioink were then measured with respect to extrudability, strength, and stiffness as determined by dynamic mechanical analysis (DMA). The DoE demonstrated that mechanical integrity increased with increasing alginate concentration. In contrast, fibrinogen and nanofibril concentration had no statistically significant effect. The optimized ink containing fibroblasts was printable using multiple nozzle sizes while also supporting fibroblast cell viability. DMA characterization further showed that the composition of the cell culture medium did not modulate the degradation rate of the hydrogel. Ultimately, the study outlines a methodology for formulating a bioink that will result in robust bioscaffolds forin vitromodel development.

Wearable Sensor System for Detection of Lactate in Sweat.

Increased development of wearable sensors for physiological monitoring has spurred complementary interest in the detection of biochemical indicators of health and performance. We report a wearable sensor system for non-invasive detection of excreted human biomarkers in sweat. The system consists of a thin, flexible, kapton patch (2.5 × 7.5 cm) that can be coated with adhesive and affixed to the skin. The system can be controlled by a cell phone via a near-field communications protocol, charged wirelessly, and the data can be downloaded and displayed in a smart phone app. The system is designed such that the sensing element plugs into a low-profile socket, and can easily be removed and replaced as needed due to saturation or aging effects. As a demonstration case, we examined using an organic electrochemical transistor (OECT) within this system to monitor lactate concentration. Several different methods for optimizing the sensor performance were compared, including altering electrode materials, employing various immobilization techniques, and tailoring operating voltages. Resulting functional response of the lactate oxidase enzyme was compared as a function of the sensor variables. The OECT sensor was shown to have high sensitivity to lactate, however the sensing range is limited to lactate concentrations below approximately 1 mM.

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Exploring the boundaries of additivity: mixtures of NADH: quinone oxidoreductase inhibitors.

The activity of mitochondrial complex I of the electron transport chain (ETC) is known to be affected by an extraordinarily large number of diverse xenobiotics, and dysfunction at complex I has been associated with a variety of disparate human diseases, including those with potentially environmentally relevant etiologies. However, the risks associated with mixtures of complex I inhibitors have not been fully explored, and this warrants further examination of potentially greater than additive effects that could lead to toxicity. A potential complication for the prediction of mixture effects arises because mammalian mitochondrial complex I has been shown to exist in two distinct dynamic conformations based upon substrate availability. In this study, we tested the accepted models of additivity as applied to mixtures of rotenone, deguelin, and pyridaben, with and without substrate limitation. These compounds represent both natural and synthetic inhibitors of complex I of the ETC, and experimental evidence to date indicates that these inhibitors share a common binding domain with partially overlapping binding sites. Therefore, we hypothesized that prediction of their mixtures effects would follow dose addition. Using human hepatocytes, we analyzed the effects of these mixtures at doses between 0.001 and 100 µM on overall cellular viability. Analysis of the dose-response curves resulting from challenge with all possible binary and ternary mixtures revealed that the appropriate model was not clear. All of the mixtures tested were found to be in agreement with response addition, but only rotenone plus deguelin and the ternary mixture followed dose addition. To determine if conformational regulation via substrate limitation could improve model selection and our predictions, we tested the models of additivity for the binary and ternary mixtures of inhibitors when coexposed with 2-deoxy-d-glucose (2-DG), which limits NADH via upstream inhibition of glycolysis. Coexposure of inhibitors with 2-DG did facilitate model selection: Rotenone plus pyridaben and the ternary mixture were in sole agreement with dose addition, while deguelin plus pyridaben was in sole agreement with response addition. The only ambiguous result was the agreement of both models with the mixture of rotenone plus deguelin with 2-DG, which may be explained by deguelin's well-known affinity for protein kinase B (Akt) in addition to complex I. Thus, our findings indicate that predictive models for mixtures of mitochondrial complex I inhibitors appear to be compound specific, and our research highlights the need to control for dynamic conformational changes to improve our mechanistic understanding of additivity with these inhibitors.

Effect of gonococcal lipooligosaccharide variation on human monocytic cytokine profile.

BACKGROUND: Neisseria gonorrhoeae is an obligate human pathogen that causes significant worldwide morbidity. N. gonorrhoeae expresses lipooligosaccharide (LOS), a phase variable molecule that plays an important role during pathogenesis of the organism. Alteration in the structure of gonococcal LOS correlates with altered disease presentation. In addition, LOS sialylation occurs readily in vivo, though the role of this sialylation during disease is unknown. RESULTS: Challenge of human monocytes with purified LOS preparations isolated from strains expressing distinct structurally defined LOSs resulted in identical production of the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-12 (IL-12). Similar results were seen when monocytes were challenged with either live or gentamicin-killed whole cell gonococcal variants expressing these LOS structures, although greater cytokine production was observed in comparison with challenge by purified LOS. Challenge of a human primary monocyte model with distinct LOS variants resulted in similar production of TNFalpha, IL-12, interleukin-10 (IL-10), and interleukin-8 (IL-8). A cytokine array was employed to allow measurement of a broad range of cytokines in samples challenge with gonococcal LOS variants as well as variants expressing sialylated LOS. Challenge of primary monocytes with sialylated gonococci was shown to elicit the production of more MCP-2 (monocyte chemoattractant protein-2) in comparison with challenge by unsialylated gonococci. CONCLUSION: We demonstrated that while alterations in the carbohydrate moiety of LOS do not impact the production of most cytokines by human monocytes, whole-cell bacterial challenge is more stimulatory than challenge with purified LOS, implying that other gonococcal cell surface antigens are important for the elicitation of cytokines. Challenge with gonococci expressing sialylated LOS resulted in elicitation of more of the chemokine MCP-2 from challenged cells in comparison with gonococci expressing unsialylated LOS. As MCP-2 is an important chemoattractant, this indicates that in vivo sialylation may play an important role during the pathogenesis of N. gonorrhoeae.

TNF-alpha-independent IL-8 expression: alterations in bacterial challenge dose cause differential human monocytic cytokine response.

We examined the effects of different bacterial doses of Neisseria gonorrhoeae on the cytokine response of primary human monocytes. The data indicate that a low multiplicity of infection (MOI) challenge (MOI = 0.1) results in substantial production of IL-8 and other chemokines/cytokines, in the absence of significant TNF-alpha production. Positive control challenges (MOI = 10) induced levels of IL-8 that were comparable to the low MOI challenges, but now induced significant levels of TNF-alpha. Induction of IL-8 expression in low MOI challenges was not mediated by an autocrine response as pretreatment of monocytes with neutralizing Abs against TNF-alpha or IL-1beta had no effect on IL-8 expression. IL-8 induction resulting from gonococcal challenge was shown to require NF-kappaB activation, though this activation was limited by the inoculating dose. These data indicate that IL-8 induction results from direct contact between bacteria and monocytes. Analysis of the overall cytokine profile revealed patterns of expression for growth-regulated oncogene, MCP-1, and IL-6 that were similar to IL-8. Analysis of various MAPKs indicated that low MOI challenges were able to efficiently activate both the ERK and p38 pathways, but in contrast to positive control samples, failed to activate the JNK pathway. A lack of phosphorylated JNK leads to decreased production of AP-1 dimers, transcription factors that are critical for efficient transcription of TNF-alpha. Therefore, we propose a mechanism where a low MOI gonococcal challenge results in diminished AP-1 activity and TNF-alpha production while IL-8 levels remain constant.