Preparation and characterization of protein-nanotube conjugates

This chapter describes methods of immobilizing proteins on carbon nanotubes, using two different routes—physical adsorption and covalent attachment. We also provide an overview on how such conjugates can be characterized with the help of various techniques, such as Raman, Fourier transform infrared (FT-IR), circular dichroism (CD), and fluorescence spectroscopies, in addition to the standard enzyme kinetic analyses of activity and stability. Both the attachment routes—covalent and noncovalent—could be used to prepare protein conjugates that retained a significant fraction of their native structure and function; furthermore, the protein conjugates were operationally stable, reusable, and functional even under harsh denaturing conditions. These studies therefore corroborate the use of these immobilization methods to engineer functional carbon nanotube-protein hybrids that are highly active and stable

Isolation and co-culture of rat parenchymal and non-parenchymal liver cells to evaluate cellular interactions and response

The liver is a central organ in the human body, and first line of defense between host and external environment. Liver response to any external perturbation is a collective reaction of resident liver cells. Most of the current in vitro liver models focus on hepatocytes, the primary metabolic component, omitting interactions and cues from surrounding environment and non-parenchymal cells (NPCs). Recent studies suggest that contributions of NPCs are vital, particularly in disease conditions, and outcomes of drugs and their metabolites. Along with hepatocytes, NPCs–Kupffer (KC), sinusoidal endothelial (LSEC) and stellate cells (SC) are major cellular components of the liver. Incorporation of primary cells in in vitro liver platforms is essential to emulate the functions of the liver, and its overall response. Herein, we isolate individual NPC cell fractions from rat livers and co-culture them in a transwell format incorporating primary rat hepatocytes with LSECs, SCs, and KCs. Our results indicate that the presence and contributions of multiple cells within the co-culture capture the interactions between hepatocytes and NPC, and modulates the responses to inflammatory stimulus such as LPS. The isolation and co-culture methods could provide a stable platform for creating in vitro liver models that provide defined functionality beyond hepatocytes alone

The protein–nanomaterial interface

Developments in the past few years have illustrated the potentially revolutionizing impact of nanomaterials, especially in biomedical imaging, drug delivery, biosensing and the design of functional nanocomposites. Methods to effectively interface proteins with nanomaterials for realizing these applications continue to evolve. Proteins are being used to control both the synthesis and assembly of nanomaterials. There has also been an increasing interest in understanding the influence of nanomaterials on the structure and function of proteins. Understanding and controlling the protein–nanomaterial interface will be crucial for designing functional protein–nanomaterial conjugates and assemblies

Engineered Trehalose Permeable to Mammalian Cells

Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies

Structure, Function, and Stability of Enzymes Covalently Attached to Single-Walled Carbon Nanotubes

We describe the structure, activity, and stability of enzymes covalently attached to single-walled carbon nanotubes (SWNTs). Conjugates of SWNTs with three functionally unrelated enzymeshorseradish peroxidase, subtilisin Carlsberg, and chicken egg white lysozymewere found to be soluble in aqueous solutions. Furthermore, characterization of the secondary and tertiary structure of the immobilized proteins by circular dichroism and fluorescence spectroscopies, respectively, and determination of enzyme kinetics revealed that the enzymes retained a high fraction of their native structure and activity upon attachment to SWNTs. The SWNT−enzyme conjugates were also more stable in guanidine hydrochloride (GdnHCl) and at elevated temperatures relative to their solution counterparts. Thus, these protein conjugates represent novel preparations that possess the attributes of both soluble enzymeshigh activity and low diffusional resistanceand immobilized enzymeshigh stabilitymaking them attractive choices for applications ranging from diagnostics and sensing to drug delivery

Protein-Carbon Nanotube Conjugates

Proteins have been conjugated to carbon nanotubes for applications in biosensing, biorecognition, delivery, and functional composites. Despite the growing interest in these carbon nanotube-protein hybrids, very little is known about how carbon nanotubes affect the structure and function of bound proteins. Here we provide an overview of our recent efforts to gain a more fundamental understanding of how proteins interact with carbon nanotubes. We also discuss recent results from our laboratories which suggest several new opportunities for protein-carbon nanotube conjugates to address problems in materials science and biotechnology

Metabolomic Modularity Analysis (MMA) to Quantify Human Liver Perfusion Dynamics

Large-scale -omics data are now ubiquitously utilized to capture and interpret global responses to perturbations in biological systems, such as the impact of disease states on cells, tissues, and whole organs. Metabolomics data, in particular, are difficult to interpret for providing physiological insight because predefined biochemical pathways used for analysis are inherently biased and fail to capture more complex network interactions that span multiple canonical pathways. In this study, we introduce a nov-el approach coined Metabolomic Modularity Analysis (MMA) as a graph-based algorithm to systematically identify metabolic modules of reactions enriched with metabolites flagged to be statistically significant. A defining feature of the algorithm is its ability to determine modularity that highlights interactions between reactions mediated by the production and consumption of cofactors and other hub metabolites. As a case study, we evaluated the metabolic dynamics of discarded human livers using time-course metabolomics data and MMA to identify modules that explain the observed physiological changes leading to liver recovery during subnormothermic machine perfusion (SNMP). MMA was performed on a large scale liver-specific human metabolic network that was weighted based on metabolomics data and identified cofactor-mediated modules that would not have been discovered by traditional metabolic pathway analyses

Best fit model parameters.

<p>Best fit model parameters.</p