Neuroprotection by chitosan nanoparticles in oxidative stress-mediated injury.

OBJECTIVE: Oxidative stress is a critical component of nervous system secondary injury. Oxidative stress produces toxic chemical byproducts including reactive aldehydes that traverse intact membranes and attack neighboring healthy cells. This secondary damage often leads to further patho-biochemical cascades that exacerbate the original insult. In this work, we investigate the therapeutic effects of chitosan nanoparticles on cell cultures exposed to oxidative stress. RESULTS: We found chitosan nanoparticles can rescue BV-2 glial cells from death, but only for cells undergoing necrosis. Necrosis occurred when cultures were challenged with high concentrations of H2O2 (> 110 µM) whereas a slow and progressive loss of cultures was observed in more dilute (50-100 µM) peroxide applications. In the latter case, the primary mode of cell death was apoptosis. These studies revealed that while rescue of H2O2 challenged cultures was achieved for necrotic cell death, no such sparing was observed in apoptotic cells. Based on the current and cumulative data regarding the membrane fusogenic properties of chitosan, we conclude that chitosan neuroprotection arises from its membrane sealing effects. Consistent with this hypothesis is the observation that apoptotic cells did not exhibit early stage membrane damage. These in vitro results elucidate mechanisms by which membrane fusogens may provide therapeutic benefit.

Remote-controlled eradication of astrogliosis in spinal cord injury via electromagnetically-induced dexamethasone release from "smart" nanowires.

We describe a system to deliver drugs to selected tissues continuously, if required, for weeks. Drugs can be released remotely inside the small animals using pre-implanted, novel vertically aligned electromagnetically-sensitive polypyrrole nanowires (PpyNWs). Approximately 1-2mm(2) dexamethasone (DEX) doped PpyNWs was lifted on a single drop of sterile water by surface tension, and deposited onto a spinal cord lesion in glial fibrillary acidic protein-luc transgenic mice (GFAP-luc mice). Overexpression of GFAP is an indicator of astrogliosis/neuroinflammation in CNS injury. The corticosteroid DEX, a powerful ameliorator of inflammation, was released from the polymer by external application of an electromagnetic field for 2h/day for a week. The GFAP signal, revealed by bioluminescent imaging in the living animal, was significantly reduced in treated animals. At 1week, GFAP was at the edge of detection, and in some experimental animals, completely eradicated. We conclude that the administration of drugs can be controlled locally and non-invasively, opening the door to many other known therapies, such as the cases that dexamethasone cannot be safely applied systemically in large concentrations.

Pushing the science forward: chitosan nanoparticles and functional repair of CNS tissue after spinal cord injury.

BACKGROUND: We continue our exploration of the large polysaccharide polymer Chitosan as an acute therapy for severe damage to the nervous system. We tested the action of subcutaneously injected nanoparticles (~ 100 - 200 nanometers in diameter; 1 mg per ml) against control injections (silica particle of the same size and concentration) in a standardized in vivo spinal cord injury model. These functional tests used standardized physiological measurements of evoked potentials arriving at the sensorimotor cortex subsequent to stimulation of the tibial nerve of the contralateral hindlimb. We further explored the degree of acetylation and molecular weight of chitosan on the success of sealing cell damage using specific probes of membrane integrity. RESULTS: Not one of the control group showed restored conduction of evoked potentials stimulated from the tibial nerve of the hindleg - through the lesion - and recorded at the sensorimotor cortex of the brain. Investigation if the degree of acetylation and molecular weight impacted "membrane sealing" properties of Chitosan were unsuccessful. Dye - exchange membrane probes failed to show a difference between the comparators in the function of Chitosan in ex vivo injured spinal cord tests. CONCLUSIONS: We found that Chitosan nanoparticles effectively restore nerve impulse transmission through the crushed adult guinea pig spinal cord in vivo after severe crush/compression injury. The tests of the molecular weight (MW) and degree of acetylation did not produce any improvement in Chitosan's membrane sealing properties.

Affinity for, and localization of, PEG-functionalized silica nanoparticles to sites of damage in an ex vivo spinal cord injury model.

BACKGROUND: Traumatic spinal cord injury (SCI) leads to serious neurological and functional deficits through a chain of pathophysiological events. At the molecular level, progressive damage is initially revealed by collapse of plasma membrane organization and integrity produced by breaches. Consequently, the loss of its role as a semi-permeable barrier that generally mediates the regulation and transport of ions and molecules eventually results in cell death. In previous studies, we have demonstrated the functional recovery of compromised plasma membranes can be induced by the application of the hydrophilic polymer polyethylene glycol (PEG) after both spinal and brain trauma in adult rats and guinea pigs. Additionally, efforts have been directed towards a nanoparticle-based PEG application.The in vivo and ex vivo applications of PEG-decorated silica nanoparticles following CNS injury were able to effectively and efficiently enhance resealing of damaged cell membranes. RESULTS: The possibility for selectivity of tetramethyl rhodamine-dextran (TMR) dye-doped, PEG-functionalized silica nanoparticles (TMR-PSiNPs) to damaged spinal cord was evaluated using an ex vivo model of guinea pig SCI. Crushed and nearby undamaged spinal cord tissues exhibited an obvious difference in both the imbibement and accumulation of the TMR-PSiNPs, revealing selective labeling of compression-injured tissues. CONCLUSIONS: These data show that appropriately functionalized nanoparticles can be an efficient means to both 1.) carry drugs, and 2.) apply membrane repair agents where they are needed in focally damaged nervous tissue.

Understanding secondary injury.

Secondary injury is a term applied to the destructive and self-propagating biological changes in cells and tissues that lead to their dysfunction or death over hours to weeks after the initial insult (the "primary injury"). In most contexts, the initial injury is usually mechanical. The more destructive phase of secondary injury is, however, more responsible for cell death and functional deficits. This subject is described and reviewed differently in the literature. To biomedical researchers, systemic and tissue-level changes such as hemorrhage, edema, and ischemia usually define this subject. To cell and molecular biologists, "secondary injury" refers to a series of predominately molecular events and an increasingly restricted set of aberrant biochemical pathways and products. These biochemical and ionic changes are seen to lead to death of the initially compromised cells and "healthy" cells nearby through necrosis or apoptosis. This latter process is called "bystander damage." These viewpoints have largely dominated the recent literature, especially in studies of the central nervous system (CNS), often without attempts to place the molecular events in the context of progressive systemic and tissue-level changes. Here we provide a more comprehensive and inclusive discussion of this topic.

Neuronal responses to an asymmetrical alternating current field can mimic those produced by an imposed direct current field in vitro.

The remarkable polarity-dependent growth and anatomical organization of neurons in vitro produced by imposed direct current (DC) voltage gradients (electrical fields; Ef) can be mimicked by another type of electrical cue. This is a properly structured asymmetrical alternating current (AC) electrical field (A-ACEf). Here we provide details on the construction of an AC signal generator in which all components of an AC waveform can be individually controlled. We show that 1) conventional symmetrical AC voltage gradients will not induce growth, guidance, or architectural changes in sympathetic neurons. We also provide the first qualitative and quantitative data showing that an asymmetric AC application can indeed mimic the DC response in chick sympathetic neurons and their growing neurites. This shift in orientation and neuronal anatomy requires dieback of some neurites and the extension of others to produce a preferred orientation perpendicular to the gradient of voltage. Our new results may lead to a noninvasive means to modify nerve growth and organization by magnetic inductive coupling at distance. These data also indicate the possibility of a means to mimic DC-dependent release of drugs or other biologically active molecules from electrically sensitive that can be loaded with these chemical cargos.

Strict perpendicular orientation of neural crest-derived neurons in vitro is dependent on an extracellular gradient of voltage.

We report extraordinary perpendicular orientations of neurons dependent on the presence of an external direct current (DC) voltage gradient. We chose chick dorsal root and postganglionic sympathetic neurons to evaluate. These were cultured in observation chambers in which the cells were separated from electrode products or substrate effects and maintained at 35°C. Both types of neurons showed a rapid restructuring of their anatomy. Typically, neurites that were not perpendicular to the voltage gradient were quickly resorbed into the cell body within a few minutes. Over 3-6 hr, significant new neurite growth occurred and was patterned perpendicular to the DC electrical field (Ef). This preferred asymmetry was dependent on the Ef, as was the initial retrograde degeneration of fibers. At 400-500 mV/mm, over 90% of the cells in culture assumed this orientation. Removal of the DC Ef led to a loss of the preferred orientation, with further random growth within the chambers. This is the first report of such responses in dorsal root ganglion neurons. We also used sympathetic neurons as a meaningful comparison to analyze whether there were any qualitative or quantitative differences between these two cell types of neural crest origin. We discuss the means by which these orientations were achieved.

Polymer and nano-technology applications for repair and reconstruction of the central nervous system.

The hydrophilic polymer PEG and its related derivatives, have served as therapeutic agents to reconstruct the phospholipid bilayers of damaged cell membranes by erasing defects in the plasmalemma. The special attributes of hydrophilic polymers when in contact with cell membranes have been used for several decades since these well-known properties have been exploited in the manufacture of monoclonal antibodies. However, while traditional therapeutic efforts to combat traumatic injuries of the central nervous system (CNS) have not been successful, nanotechnology-based drug delivery has become a new emerging strategy with the additional promise of targeted membrane repair. As such, this potential use of nanotechnology provides new avenues for nanomedicine that uses nanoparticles themselves as the therapeutic agent in addition to their other functionalities. Here we will specifically address new advances in experimental treatment of Spinal Cord and Traumatic Brain injury (SCI and TBI respectively). We focus on the concept of repair of the neurolemma and axolemma in the acute stage of injury, with less emphasis on the worthwhile, and voluminous, issues concerning regenerative medicine/nanomedicine. It is not that the two are mutually exclusive - they are not. However, the survival of the neuron and the tissues of white matter are critical to any further success in what will likely be a multi-component therapy for TBI and SCI. This review includes a brief explanation of the characteristics of traumatic spinal cord injury SCI, the biological basis of the injuries, and the treatment opportunities of current polymer-based therapies. In particular, we update our own progress in such applications for CNS injuries with various suggestions and discussion, primarily nanocarrier-based drug delivery systems. The application of nanoparticles as drug-delivery vehicles to the CNS may likely be advantageous over existing molecular-based therapies. As a "proof-of-concept", we will discuss the recent investigations that have preferentially facilitated repair and functional recovery from breaches in neural membranes via rapid sealing and reassembly of the compromised site with silica or chitosan nanoparticles.

Biotin-doped porous polypyrrole films for electrically controlled nanoparticle release.

A novel method for the preparation of biotin-doped porous conductive surfaces has been suggested for a variety of applications, especially for an electrically controlled release system. Well-ordered and three-dimensional porous conductive structures have been obtained by the electrochemical deposition of the aqueous biotin-pyrrole monomer mixture into particle arrays, followed by subsequent removal of the colloidal particles. Advantageously, direct incorporation of biotin molecules enhances the versatility by modifying surfaces through site-directed conjugate formation, thus facilitating further reactions. In addition, the porosity of the surfaces provides a significant impact on enhanced immobilization and efficient release of streptavidin-tagged gold nanoparticles. Biotinylated porous polypyrrole (Ppy) films were characterized by several techniques: (1) scanning electron microscopy (SEM) to evaluate surface topography, (2) X-ray photoelectron spectroscopy (XPS) to assess the potential-dependent chemical composition of the films, (3) four-point probe evaluation to measure the conductivity, cyclic voltammetry to observe surface eletroactivity, and contact angle measurement to evaluate the surface wettability, and (4) fluorescence microscopy to image and quantify the adsorption and release of gold nanoparticles. Overall, our results demonstrate that these biotinylated porous Ppy films, combined with electrical stimulation, permit a programmable release of gold nanoparticles by altering the chemical strength of the Ppy-biotin interaction.

Well-ordered porous conductive polypyrrole as a new platform for neural interfaces.

We present the preparation of electrically conductive, porous polypyrrole surfaces and demonstrate their use as an interactive substrate for neuronal growth. Nerve growth factor (NGF)-loaded porous conducting polymers were initially prepared by electrochemical deposition of a mixture of pyrrole monomers and NGF into two- or three-dimensional particle arrays followed by subsequent removal of a sacrificial template. Morphological observation by scanning electron microscopy (SEM) revealed these to possess high regularity and porosity with well-defined topographical features. A four-point probe study demonstrated remarkable electrical activities despite the presence of voids. In addition, we investigated the effects of these surfaces on cellular behaviors using PC 12 cells in the presence and absence of electrical stimulation. Our results suggest that the surface topography as well as an applied electrical field can play a crucial role in determining further cell responses. Indeed, surface-induced preferential regulation leads to enhanced cellular viability and neurite extension. Establishing the underlying cellular mechanisms in response to various external stimuli is essential in that one can elicit positive neuronal guidance and modulate their activities by engineering a series of electrical, chemical, and topographical cues.

The effect of an electrically conductive carbon nanotube/collagen composite on neurite outgrowth of PC12 cells.

We report the preparation of an electrically conductive composite composed of collagen and carbon nanotubes (CNTs) and its use as a substrate for the in vitro growth of PC12 cells. Morphological observation by scanning electron microscopy (SEM) indicated the homogenous dispersion of CNTs in the collagen matrix. Four-point probe and cyclic voltammogram studies demonstrated the enhanced electroactivity and a lowered electrical resistivity of the resulting composites even at low loadings (<5%) of CNTs. Cellular metabolic activity was evaluated by the MTT assay. Cell viability was systematically related to the amount of CNTs embedded in the collagen matrix. SEM and immunofluorescent images have indicated that the morphological features of PC12 cells were dominantly influenced by electrical potential. Greater neurite extension was preferentially induced on the exposure of electrical stimulation by facilitating the differentiation of PC12 cells into neurons indicated by more significant filopodium extension. These electrically conductive, biocompatible CNT/collagen composites could be of benefit for the development of novel neural electrodes, enhancing the growth, differentiation, and branching of neurons in an electrically driven way.

The preparation of polypyrrole surfaces in the presence of mesoporous silica nanoparticles and their biomedical applications.

The deposition of carboxylic acid-terminated conducting polymer into two- or three-dimensional structures made up of colloidal particles has been successfully completed. This was accomplished using electrochemical deposition of ordered arrays of mesoporous silica nanoparticles (MSNs) as a template. Subsequent removal of the template yielded a porous polypyrrole surface. The co-polymerization of pyrrole with carboxylic acid-terminated pyrrole derivatives overcame the limitations of a lack of reactive functional groups--by facilitating the direct coupling of the film with biomolecules or drugs on the surface. Such Ppy films were characterized by several techniques: (1) scanning electron microscope (SEM) to evaluate surface topography, (2) x-ray photoelectron spectroscopy (XPS) to assess the chemical composition of the films, (3) four-point probe to measure the conductivity, and cyclic voltammogram to observe surface electroactivity. To assay the biological effectiveness of this preparation, we used phase-contrast light microscopy to compare neurite outgrowth from PC 12 cells grown on Ppy films in the presence and absence of electrical stimulation. These electrically functional, biocompatible composites show promise as novel neural implants that would deliver specific biologically active molecules in a highly localized manner to damaged or otherwise vulnerable cells such as found in the nervous system.

Perpendicular organization of sympathetic neurons within a required physiological voltage.

In vitro, postganglionic sympathetic neurons (PSNs) profoundly organize their anatomy according to cues provided by an extracellular voltage. Over 90% of PSNs retract neurites that are parallel/tangential to a gradient of approximately 400 mV/mm. Complete neurite retraction takes approximately 20-40 minutes. Subsequently, neurites grow out from the soma, but now perpendicular to the lines of force while branching profusely. The complete restructuring of the neurons anatomy takes 2-3 hours at 35 degrees C. The maintenance of this asymmetrical anatomy requires the continuous presence of the extracellular electrical field (Ef). We discuss this observation relative to the organization of neurons residing in natural voltage gradients that exist across all epithelia in which neurons are born, mature, or migrate.

Functional silica nanoparticle-mediated neuronal membrane sealing following traumatic spinal cord injury.

The mechanical damage to neurons and their processes induced by spinal cord injury (SCI) causes a progressive cascade of pathophysiological events beginning with the derangement of ionic equilibrium and collapse of membrane permeability. This leads to a cumulative deterioration of neurons, axons, and the tissue architecture of the cord. We have previously shown that the application of the hydrophilic polymer polyethylene glycol (PEG) following spinal cord or brain injury can rapidly restore membrane integrity, reduce oxidative stress, restore impaired axonal conductivity, and mediate functional recovery in rats, guinea pigs, and dogs. However there are limits to both the concentration and the molecular weight of the application that do not permit the broadest recovery across an injured animal population. In this study, PEG-decorated silica nanoparticles (PSiNPs) sealed cells, as shown by the significantly reduced leakage of lactate dehydrogenase from damaged cells compared with uncoated particles or PEG alone. Further in vivo tests showed that PSiNPs also significantly reduced the formation of reactive oxygen species and the process of lipid peroxidation of the membrane. Fabrication of PSiNPs containing embedded dyes also revealed targeting of the particles to damaged, but not undamaged, spinal cord tissues. In an in vivo crush/contusion model of guinea pig SCI, every animal but one injected with PSiNPs recovered conduction through the cord lesion, whereas none of the control animals did. These findings suggest that the use of multifunctional nanoparticles may offer a novel treatment approach for spinal cord injury, traumatic brain injury, and possibly neurodegenerative disorders.

Neuroprotection from secondary injury by polyethylene glycol requires its internalization.

Polyethylene glycol (PEG) is well known to both fuse and repair cell membranes. This capability has been exploited for such diverse usages as the construction of hybridomas and as a reparative agent following neurotrauma. The latter development has proceeded through preclinical testing in cases of naturally induced paraplegia in dogs. The mechanisms of action of polymer-mediated neurorepair/neuroprotection are still under investigation. It is likely that the unique interaction of hydrophilic polymers with the mechanical properties of cell membranes in concert with an ability to interfere with mechanisms of secondary injury such as the production of highly reactive oxygen species (ROS or ;free radicals') is the basis for neuroprotection by polymers. Here we provide further evidence that the ability of PEG to reduce or limit secondary injury and/or lipid peroxidation (LPO) of membranes requires entry of PEG into the cytosol, further suggesting a physical interaction with the membranes of organelles such as mitochondria as the initial event leading to neurorepair/neuroprotection. We have evaluated this relationship in vitro using acrolein, a potent endogenous toxin that is a product of LPO. Acrolein can pass through cell membranes with ease, inducing progressive LPO in ;bystander' cells, and the production of even more acrolein by inducing its own production. Immediate application of PEG (10 mmol l(-1), 2000 Da) to poisoned neurons in vitro was unable to rescue them from necrosis and death. Furthermore, three-dimensional confocal microscopy of fluorescently decorated PEG shows that it does not enter these cells for up to 2 h after application. By this time the mechanisms of necrosis are likely irreversible. Additionally, severe oxygen and or glucose deprivation of spinal cord white matter in vitro also initiates LPO. Addition of potent free radical scavengers such as ascorbic acid or superoxide dismutase (SOD) is able to interfere with this process, but PEG is not. Taken together, these data are consistent with the hypothesis that PEG is able to rescue mechanically damaged cells, based on a restructuring of the damaged plasmalemma. Furthermore, in compromised cells with an intact cell membrane, PEG must first gain access to the cytosol where this same capability may be useful in restoring the integrity of cellular organelles such as mitochondria, though the intracellular concentration of the polymer must be significant relative to the concentration of toxins produced by LPO in order to rescue the cell.

Hydralazine rescues PC12 cells from acrolein-mediated death.

Acrolein, a major lipid peroxidation product, has been associated with both CNS trauma and neurodegenerative diseases. Because of its long half-life, acrolein is a potent endogenous toxin capable of killing healthy cells during the secondary injury process. Traditionally, attempts to intervene in the process of progressive cell death after the primary injury have included scavenging reactive oxygen species (so-called free radicals). The animal data supporting such an approach have generally been positive, but all human clinical trials attempting a similar outcome in human CNS injury have failed. New drugs that might reduce toxicity by scavenging the products of lipid peroxidation present a promising, and little investigated, therapeutic approach. Hydralazine, a well-known treatment for hypertension, has been reported to react with acrolein, forming hydrazone in cell-free systems. In the companion paper, we have established an acrolein-mediated cell injury model using PC12 cells in vitro. Here we test the hypothesis that the formation of hydrazone adducts with acrolein is able to reduce acrolein toxicity and spare a significant percentage of the population of PC12 cells from death. Concentrations of approximately 1 mM of this aldehyde scavenger can rescue over 80% of the population of PC12 cells. This study provides a basis for a new pharmacological treatment to reduce the effects of secondary injury in the damaged and/or diseased nervous system. In particular, we describe the need for new drugs that possess aldehyde scavenging properties but do not interfere with the regulation of blood pressure.

Acrolein-mediated mechanisms of neuronal death.

It is well known that traumatic injury in the central nervous system can be viewed as a primary injury and a secondary injury. Increases in oxidative stress lead to breakdown of membrane lipids (lipid peroxidation) during secondary injury. Acrolein, an alpha,beta-unsaturated aldehyde, together with other aldehydes, increases as a result of self-propagating lipid peroxidation. Historically, most research on the pathology of secondary injury has focused on reactive oxygen species (ROS) rather than lipid peroxidation products. Little is known about the toxicology and cell death mediated by these aldehydes. In this study, we investigated and characterized certain features of cell death induced by acrolein on PC12 cells as well as cells from dorsal root ganglion (DRG) and sympathetic ganglion in vitro. In the companion paper, we evaluated a possible means to interfere with this toxicity by application of a compound that can bind to and inactivate acrolein. Here we use both light and atomic force microscopy to study cell morphology after exposure to acrolein. Administration of 100 microM acrolein caused a dramatic change in cell morphology as early as 4 hr. Cytoskeletal structures significantly deteriorated after exposure to 100 microM acrolein as demonstrated by fluorescence microscopy, whereas calpain activity increased significantly at this concentration. Cell viability assays indicated significant cell death with 100 microM acrolein by 4 hr. Caspase 3 activity and DNA fragmentation assays were performed and supported the notion that 100 microM acrolein induced PC12 cell death by the mechanism of necrosis, not apoptosis.

Three-dimensional imaging of living and dying neurons with atomic force microscopy.

Atomic Force Microscopy (AFM) has been used to image the morphology of developing neurons and their processes. Additionally, AFM can physically interact with the cell under investigation in numerous ways. Here we use the AFM to both three-dimensionally image the neuron and to inflict a nano/micro-puncture to its membrane. Thus, the same instrument used as a tool to precisely penetrate/cut the membrane at the nanoscale level is employed to image the morphological responses to damage. These first high resolution AFM images of living chick dorsal root ganglion cells and cells of sympathetic ganglion and their growing processes provide confirmation of familiar morphologies. The increased resolution of the AFM revealed these structures to be significantly more complex and variable than anticipated. Moreover we describe novel, dynamic, and unreported architectures, particularly large dorsally projecting ridges, spines, and ribbons of cytoplasm that appear and disappear on the order of minutes. In addition, minute (ca. 100 nm) hair-like extensions of membrane along the walls of nerve processes that also shift in shape and density, appearing and disappearing over periods of minutes were seen. We also provide "real time" images of the death of the neuron cell body after nano/micro scale damage to its membrane. These somas excreted their degraded cytoplasm, revealed as an enlarging pool beneath and around the cell. Conversely, identical injury, even repeated perforations and nanoslices, to the neurite's membrane do not lead to demise of the process. This experimental study not only provides unreported neurobiology and neurotrauma, but also emphasizes the unique versatility of AFM as an instrument that can (1) physically manipulate cells, (2) provide precise quantitative measurements of distance, surface area and volume at the nanoscale if required, (3) derive physiologically significant data such as membrane pressure and compliance, and (4) during the same period of study--provide unexcelled imaging of living samples.

Restoring function to the injured human spinal cord.

Of catastrophic traumas to the human body, spinal cord injury (SCI) has least benefited innovations arising from the new biology. Since after WW II, the "standard of care" for SCI has changed little. The controversial use of high dosages of steroids has provided only modest benefit to patients--but not without the enhanced risk of mortality. Novel therapies arising from biochemistry and genetics have not materialized in over 15 years, and are unlikely to in the author's opinion. Instead, appreciation of biophysics and cell physiology in controlling nerve injury, growth, regeneration, and function has produced innovative clinical approaches now in testing in human spinal cord injury.