Spinal cord injury: a review of current therapy, future treatments, and basic science frontiers.

The incidence of acute and chronic spinal cord injury (SCI) in the United States is more than 10,000 per year, resulting in 720 cases per million persons enduring permanent disability each year. The economic impact of SCI is estimated to be more than 4 billion dollars annually. Preclinical studies, case reports, and small clinical trials suggest that early treatment may improve neurological recovery. To date, no proven therapeutic modality exists that has demonstrated a positive effect on neurological outcome. Emerging data from recent preclinical and clinical studies offer hope for this devastating condition. This review gives an overview of current basic research and clinical studies for the treatment of SCI.

Nanofabrication of insulated scanning probes for electromechanical imaging in liquid solutions.

In this paper, the fabrication and electrical and electromechanical characterization of insulated scanning probes have been demonstrated in liquid solutions. The silicon cantilevers were sequentially coated with chromium and silicon dioxide, and the silicon dioxide was selectively etched at the tip apex using focused-electron-beam-induced etching (FEBIE) with XeF(2). The chromium layer acted not only as the conductive path from the tip, but also as an etch-resistant layer. This insulated scanning probe fabrication process is compatible with any commercial AFM tip and can be used to easily tailor the scanning probe tip properties because FEBIE does not require lithography. The suitability of the fabricated probes is demonstrated by imaging of a standard topographical calibration grid as well as piezoresponse force microscopy (PFM) and electrical measurements in ambient and liquid environments.

Enhanced stability of enzymes adsorbed onto nanoparticles.

We have discovered that the highly curved surface of C60 fullerenes enhances enzyme stability in strongly denaturing environments to a greater extent than flat supports. The half-life of a model enzyme, soybean peroxidase, adsorbed onto fullerenes at 95 degrees C was 117 min, ca. 2.5-fold higher than that of the enzyme adsorbed onto graphite flakes and ca. 13-fold higher than that of the native enzyme. Furthermore, this phenomenon is not unique to fullerenes, but can also be extended to other nanoscale supports including silica and gold nanoparticles. The enhanced stability was exploited in the preparation of highly active and stable polymer-nanocomposite films. The ability to enhance protein stability by interfacing them with nanomaterials may impact numerous fields ranging from the design of diagnostics, sensors, and nanocomposites to drug delivery.

NANOMATERIALS FOR PROTEIN MEDIATED THERAPY AND DELIVERY.

There has been a significant amount of research done on liposomes and nanoparticles as drug carriers for protein drugs. Proteins and enzymes have been used both as targeting moieties and for their therapeutic potential. High specificity and rapid reaction rates make proteins and enzymes excellent candidates for therapeutic treatment, but some limitations exist. Many of these limitations can be addressed by a well studied nanotechnology based delivery system. Such a system can provide a medium for delivery, stabilization of the drugs, and enable site specific accumulation of drugs. Nanomedicines such as these have great potential to revolutionize the pharmaceutical industry and improve healthcare worldwide.

Nanoparticles for targeted delivery of antioxidant enzymes to the brain after cerebral ischemia and reperfusion injury.

Stroke is one of the major causes of death and disability in the United States. After cerebral ischemia and reperfusion injury, the generation of reactive oxygen species (ROS) and reactive nitrogen species may contribute to the disease process through alterations in the structure of DNA, RNA, proteins, and lipids. We generated various nanoparticles (liposomes, polybutylcyanoacrylate (PBCA), or poly(lactide-co-glycolide) (PLGA)) that contained active superoxide dismutase (SOD) enzyme (4,000 to 20,000 U/kg) in the mouse model of cerebral ischemia and reperfusion injury to determine the impact of these molecules. In addition, the nanoparticles were untagged or tagged with nonselective antibodies or antibodies directed against the N-methyl-D-aspartate (NMDA) receptor 1. The nanoparticles containing SOD protected primary neurons in vitro from oxygen-glucose deprivation (OGD) and limited the extent of apoptosis. The nanoparticles showed protection against ischemia and reperfusion injury when applied after injury with a 50% to 60% reduction in infarct volume, reduced inflammatory markers, and improved behavior in vivo. The targeted nanoparticles not only showed enhanced protection but also showed localization to the CA regions of the hippocampus. Nanoparticles alone were not effective in reducing infarct volume. These studies show that targeted nanoparticles containing protective factors may be viable candidates for the treatment of stroke.

Enzyme-nanoparticle conjugates for biomedical applications.

Enzymes hold a great promise as therapeutic agents because of their unique specificity and high level of activity. Yet, clinically important enzyme drugs are for less common than conventional low molecular weight drugs due to a number of disadvantages. Most important among these are poor stability, potential immunogenicity, and potential systemic toxicity. Recent developments in synthesis and characterization of nanoparticles and exciting novel properties of some classes of nanomaterials have boosted interest in the potential use of nanoparticles as carriers of enzyme drugs. In certain cases, use of enzymes attached to nanoparticles can help to overcome some of the above problems and improve the prospects of clinical applications of enzyme drugs. Here, we review recent data on the use of nanoparticles as carriers for several clinically important enzyme drugs and discuss advantages and potential limitations of such constructs. While promising preliminary results were obtained with regard to their performance in vitro and in some animal models, further investigations and clinical trials, as well as addressing regulatory issues, are warranted to make these delivery systems suitable for clinical applications.

Role of cytoskeletal components in stress-relaxation behavior of adherent vascular smooth muscle cells.

A number of recent studies have demonstrated the effectiveness of atomic force microscopy (AFM) for characterization of cellular stress-relaxation behavior. However, this technique's recent development creates considerable need for exploration of appropriate mechanical models for analysis of the resultant data and of the roles of various cytoskeletal components responsible for governing stress-relaxation behavior. The viscoelastic properties of vascular smooth muscle cells (VSMCs) are of particular interest due to their role in the development of vascular diseases, including atherosclerosis and restenosis. Various cytoskeletal agents, including cytochalasin D, jasplakinolide, paclitaxel, and nocodazole, were used to alter the cytoskeletal architecture of the VSMCs. Stress-relaxation experiments were performed on the VSMCs using AFM. The quasilinear viscoelastic (QLV) reduced-relaxation function, as well as a simple power-law model, and the standard linear solid (SLS) model, were fitted to the resultant stress-relaxation data. Actin depolymerization via cytochalasin D resulted in significant increases in both rate of relaxation and percentage of relaxation; actin stabilization via jasplakinolide did not affect stress-relaxation behavior. Microtubule depolymerization via nocodazole resulted in nonsignificant increases in rate and percentage of relaxation, while microtubule stabilization via paclitaxel caused significant decreases in both rate and percentage of relaxation. Both the QLV reduced-relaxation function and the power-law model provided excellent fits to the data (R(2)=0.98), while the SLS model was less adequate (R(2)=0.91). Data from the current study indicate the important role of not only actin, but also microtubules, in governing VSMC viscoelastic behavior. Excellent fits to the data show potential for future use of both the QLV reduced-relaxation function and power-law models in conjunction with AFM stress-relaxation experiments.

Cigarette smoke extract stimulates interleukin-8 production in human airway epithelium and is attenuated by superoxide dismutase in vitro.

BACKGROUND: Cigarette smoke exposure (CSE) results in extensive inflammation in the upper and lower airways. Reactive oxygen species, such as superoxide, have been shown to be potent mediators of this inflammation. METHODS: Mucosal biopsy specimens were collected from patients undergoing sinonasal surgery and were used as a source of primary epithelial cells. Human sinonasal epithelial (HSNE) cells and were isolated from sinus tissue, maintained in culture, and ultimately treated with varying concentrations of CSE with or without free superoxide dismutase (SOD). Supernatants and cell lysates were examined for the proinflammatory cytokine interleukin (IL)-8. Similar experiments were performed using normal human bronchial epithelial (NHBE) cell lines. RESULTS: CSE induces both secretion and intracellular production of the proinflammatory cytokine IL-8 by HSNE cells in a dose-dependent manner. Furthermore, this up-regulation can be suppressed by SOD. CSE induces secretion of IL-8 in NHBEs that is also suppressed by SOD. CONCLUSION: Inflammation in the airway after CSE can be blocked by SOD in this in vitro model. The ability to attenuate CSE-induced inflammation with SOD could provide a therapeutic/preventative approach for individuals with cigarette smoke exposure.

Towards local electromechanical probing of cellular and biomolecular systems in a liquid environment.

Electromechanical coupling is ubiquitous in biological systems, with examples ranging from simple piezoelectricity in calcified and connective tissues to voltage-gated ion channels, energy storage in mitochondria, and electromechanical activity in cardiac myocytes and outer hair cell stereocilia. Piezoresponse force microscopy (PFM) originally emerged as a technique to study electromechanical phenomena in ferroelectric materials, and in recent years has been employed to study a broad range of non-ferroelectric polar materials, including piezoelectric biomaterials. At the same time, the technique has been extended from ambient to liquid imaging on model ferroelectric systems. Here, we present results on local electromechanical probing of several model cellular and biomolecular systems, including insulin and lysozyme amyloid fibrils, breast adenocarcinoma cells, and bacteriorhodopsin in a liquid environment. The specific features of PFM operation in liquid are delineated and bottlenecks on the route towards nanometre-resolution electromechanical imaging of biological systems are identified.

Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme.

Adsorption of chicken egg lysozyme on silica nanoparticles of various diameters has been studied. Special attention has been paid to the effect of nanoparticle size on the structure and function of the adsorbed protein molecules. Both adsorption patterns and protein structure and function are strongly dependent on the size of the nanoparticles. Formation of molecular complexes is observed for adsorption onto 4-nm silica. True adsorptive behavior is evident on 20- and 100-nm particles, with the former resulting in monolayer adsorption and the latter yielding multilayer adsorption. A decrease in the solution pH results in a decrease in lysozyme adsorption. A change of protein structure upon adsorption is observed, as characterized by a loss in alpha-helix content, and this is strongly dependent on the size of the nanoparticle and the solution pH. Generally, greater loss of alpha helicity was observed for the lysozyme adsorbed onto larger nanoparticles under otherwise similar conditions. The activity of lysozyme adsorbed onto silica nanoparticles is lower than that of the free protein, and the fraction of activity lost correlates well with the decrease in alpha-helix content. These results indicate that the size of the nanoparticle, perhaps because of the contributions of surface curvature, influences adsorbed protein structure and function.

Structure and function of enzymes adsorbed onto single-walled carbon nanotubes.

We have examined the structure and function of two enzymes, alpha-chymotrypsin (CT) and soybean peroxidase (SBP), adsorbed onto single-walled carbon nanotubes (SWNTs). SBP retained up to 30% of its native activity upon adsorption, while the adsorbed CT retained only 1% of its native activity. Analysis of the secondary structure of the proteins via FT-IR spectroscopy revealed that both enzymes undergo structural changes upon adsorption, with substantial secondary structural perturbation observed for CT. Consistent with these results, AFM images of the adsorbed enzymes indicated that SBP retains its native three-dimensional shape while CT appears to unfold on the SWNT surface. This study represents the first in depth investigation of protein structure and function on carbon nanotubes, which is critical in designing optimal carbon nanotube-protein conjugates.