Faculty Directory

Research of our group is broadly related to areas of Bionanotechnology and Biomedical Imaging. The first group of projects focuses on design of biomedical nanodevices based on simultaneous attachment of two or more functional proteins to the same nanoparticle. The second group of projects is devoted to development of advanced scanning probe microscopy methods for imaging of biological objects in native-like environment.

Before complex conjugates consisting of two or more functional enzymes attached to a single nanoparticle can be fabricated, it is necessary to accumulate fundamental knowledge about the building blocks of such conjugates. We study how attachment of protein to a nanoparticle affects its structure and function¹. Our goal is to achieve control over the activity of enzymes in their conjugates with nanoparticles. Variation of such parameters as nanoparticle size, spacing of the proteins on the nanoparticle surface, and length of the molecular chains used to tether the proteins to the nanoparticle can be effectively used to optimize enzymatic activity.

Use of enzymatic reactions in non-aqueous media is a promising method for synthesis of many complex organic molecules due to high selectivity, stereospecificity of enzymes, and the possibility to carry out reactions in mild conditions. Immobilization of enzymes on a solid support is broadly used in nonaqueous biocatalysis to enhance their stability and allow easy extraction of the enzyme from the reaction medium. Although enzymes exhibit considerable activity in non-aqueous media, the activity is low compared to that in water. Immobilization can result in further decrease of enzymatic activity because of the interaction of protein molecules with the solid surface. For reactions in aqueous solutions, it is known that enzymes immobilized on nanoparticles generally show higher activity than those immobilized on conventional surfaces. Here we propose to study enzymatic catalysis in non-aqueous media for model protein immobilized on nanoparticles. Our goal is to achieve fundamental understanding of the effect of such immobilization on enzyme structure and function. Specifically, we will study effects of the nanoparticle material (hydrophilic or hydrophobic), immobilization method (adsorption or covalent binding), and nanoparticle size on conformation and activity of a model enzyme, subtilisin Carlsberg, in nonaqueous media.

Membrane-associated proteins lose most or all of their native activity when used in aqueous solutions in the absence of cell membranes or surfactants. Incorporation into liposomes can sometimes be used to provide native-like conditions for membrane proteins, but low stability of liposomes limits their applications. Conjugation to nanoparticles, especially to those possessing both hydrophobic and hydrophilic areas on the surface, is expected to stabilize these proteins and make possible their use in the absence of cell membranes. We plan to study interaction of membrane proteins with mesoporous silica nanoparticles. The latter possess a system of highly ordered nanoscale pores filled by hydrophobic surfactant and hydrophilic silica walls. The advantage of using mesoporous silica nanoparticles is our ability to precisely control both the pore size and wall thickness, which gives rise to the possibility of tuning up the ratio between the hydrophilic and hydrophobic areas to fit a particular membrane protein.

Currently, tissue plamionogen activator (tPA) is used clinically in post-myocardial infarction treatment to inhibit blood clot formation. tPA activates plasmin, a protease capable of dissolving blood clot components, by cleaving its inactive precursor, plasminogen. One problem associated with the clinical use of tPA is its systemic toxicity: it activates plasminogen not only in blood clots but also in the blood streamand thus giving rise to increased risk of hemorrage. Creation of a more selective form of tPA that would specifically bind to blood clots and only activate plasmin in their vicinity is thus an important therapeutic problem solving which can decrease systemic toxicity of tPA and reduce mortality associated with its clinical application.

Our research goal is development of a nanodevice based on simultaneous attachment of tPA and antrifibrin antbody to polylactide nanoparticles to selectively deliver tPA to blood clots through fibrin-specific antibody binding (Figure 1). Such protein-nanoparticle conjuates can be advantageous over other delivery systems because of relative simplicity of their preparation, higher stability, and possibility to regulate their properties by optimizing the ratios between the functional components and changing the nanoparticle propeties.

We first plan to study fundamental aspects of simultaneous conjugation of two proteins to polylactide nanoparticles. Several attachment strategies will be compared. Possibility to control system's bioactivity and fibrin-binding ability by varying the number of protein molecules of each type attached to a nanoparticle will be studied. Nanoparticle size and surface charge density, as well as the length of the spacer will also be used to tune up the properties of the conjugates. In vitro characterization experiments with artificial fibrin clots will be used to obtain then systems with optimal targeting efficiency and clot dissolution rate.

At the final stages of the project, clearance rate for protein-nanoparticle conjugates will be assessed in vivo in Sprague-Dawley rats. Rat thrombosis model will be used for in vivo evaluation of the therapeutic effectiveness of the proposed nanodevices.

The outcome of spinal cord injury depends on the extent of secondary damage produced by a series of cellular and molecular events initiated by the primary trauma. Secondary injury is a combination of several factors contributing to cell death, including glutamatergic excitotoxicity, free-radical damage, cytokines, and inflammation. Since secondary injury is a multi-dimensional disorder resulting from numerous interdependent molecular and biochemical events, its treatment involves a number of challenges. Current therapeutic strategies include the use of anti-apoptotic drugs, free radical scavengers, and anti-inflammatory agents. Each of these approaches focuses on only one aspect of the entire process. Thus, a need exists for the development of innovative multitask strategies, which would be able to block key reactions in cellular and molecular injury cascades, thus reducing secondary tissue damage, minimizing side effects, and improving functional recovery.

Applications of nanotechnology in basic and clinical neuroscience are in only the early stages of development. One significant achievement was demonstration that functionalized polybutylcyanoacrylate (PBCA) nanoparticles can penetrate the blood-brain barrier and be used for drug and gene delivery to the central nervous system (CNS). This finding, in combination with our approach of conjugating several functional enzyme molecules to polymeric nanoparticles, provides a platform for both delivery of therapeutic agents to CNS and creation of multitask therapeutic devices. In our first prototypical systems, the enzymes will be chosen to simultaneously address two different aspects of secondary injury and will include glutamate receptor ligand peptides (anti-apoptosis) and free radical scavengers, such as superoxide dismutase (SOD) and catalase (anti-oxidative injury). The effectiveness of the nanodevices in preventing glutamate-induced neurotoxicity and free-radical injury will be evaluated using chicken and rat neuronal-cell culture models.

Figure 2 shows a representative image of a chicken neuron treated by nanoparticles that carry covalently attached superoxide dismutase and anti-NR1 glutamate receptor antibody. Green fluorescence indicates strong binding of the nanoparticles to the cells. In vitro studies of therapeutic effect of such nanodevices on neurons treated by reactive oxygen species and glutamate, as well as cytotoxicity studies are currently in progress. This research is supported by South Carolina Spinal Cord Injury Research Fund.

Antibiotics once were regarded as a universal antimicrobial weapon. However, many bacteria have developed antibiotic-resistant strains, and the number of such resistant species is growing quickly. Alternative approaches to treat bacterial infections are urgently needed in healthcare facilities worldwide. Use of antimicrobial peptides for this purpose has recently been studied extensively. Here, we propose to use simultaneous covalent attachment of several antimicrobial peptides and a targeting antibody to a polymeric nanoparticle to create multifunctional antibacterial nanodevices. This project will focus on design of nanodevices for treatment of Pseudomonas Aeruginosa infection. P. Aeruginosa is the most common opportunistic pathogen known to produce antibiotic-resistant strains quickly. Extremely rare in healthy people, P. aeruginosa infections often occur in immunocompromised patients. P. Aeruginosa infection is a serious problem in patients hospitalized with burns, cancer, and cystic fibrosis. The case fatality rate in these patients is 50 percent.

In preliminary studies,² we found that covalent attachment of hen-egg lysozyme to positively charged polystyrene latex nanoparticles significantly enhances its antibacterial activity against model gram-positive and gram-negative bacteria, probably due to better targeting by positively charged nanoparticles (Figure 3). Here, we propose to expand these studies to test antibacterial activity of several natural antibacterial peptides, including lysozyme, acyloxyacyl hydrolase, human b-defensin and LL-37, attached to polymeric nanoparticles. Our first hypothesis is that targeted delivery of antimicrobial proteins attached to nanoparticles enhances their antibacterial activity against P. Aeruginosa. Our second hypothesis is that simultaneous attachment of two antibacterial peptides known to act synergistically in their free form to the same nanoparticle enhances the synergism.

The following experiments are necessary to verify these hypotheses. First, we plan to optimize the conditions for the preparation of conjugates of individual antibacterial peptides. Such parameters as nanoparticle size and material, density of surface coverage by the proteins, and presence of a targeting antibody will be used to tailor the activity of antibacterial microspheres to achieve optimal performance against P. Aeruginosa in vitro. Second, we will prepare and characterize in vitro conjugates consisting of two synergistic antimicrobial peptides simultaneously attached to the same nanoparticle. Third, the most promising systems will be tested in vivo using a mouse wound model infected with P. Aeruginosa.

Atherosclerosis, the leading cause of death in the developed world, is responsible for more than half of the yearly mortality in the United States. One of the most important risk factors for atherosclerosis is hyperlipidemia, characterized by elevated blood levels of low-density lipoproteins (LDLs). Excessive LDLs can accumulate in the vicinity of a small vascular injury, forming so-called fatty streaks, the first manifestation of atherosclerotic plaque. Hyperlipidemia is often related to the lack of LDL-receptors in hepatocytes, which consequently couldn’t recognize low-density lipoproteins and make impossible further metabolism of bad cholesterol located in LDLs. We propose to use nanoparticles to enhance delivery of low-density lipoprotein to liver. Nanoparticles are known to be actively uptaken by Kuppher cells in the liver with the half life of several minutes. We propose to use monoclonal antibody to human apolipoprotein-B100 covalently attached to biocompatible (PLGA, or polyketal) nanoparticles. After injection, such nanoparticles will adsorb LDLs via antibody-antigen interactions, and these complexes will be quickly uptaken by Kuppher cells. The latter will redirect LDLs to lysosomes, thus providing a similar pathway to that of normal uptake by LDL receptors.

Although coupling between electrical and mechanical phenomena is a universal feature of all biological systems, little is known about origins of biological electromechanical phenomena on the cellular and subcellular scale. Understanding the underlying molecular mechanisms may have tremendous impact on general understanding of biological processes and specific biomedical applications. Electromechanical stimulation of cells can become a valuable tool for their characterization and could eventually result in the development of novel therapeutic interventions.

Insufficient information about electromechanical phenomena in biological systems is a result of the lack of characterization techniques capable of providing such information on the nanometer scale and capable of operation in liquid environment. Piezoresponse force microscopy (PFM) is a scanning probe technique that enables mapping of electromechanical properties at the nanoscale. In PFM, voltage is applied to the tip and mechanical response of the sample to this voltage is registered. We demonstrated the first evidence of piezoelectricity of an individual amyloid fibril bundle using PFM (Figure 4).³ Two PFM scans with applied ac biases of 10 V (Fig. 4a,c) and 2 V (Fig. 4b,d) of the same 10 nm lysozyme fibril bundle in an aqueous environment shows similar topography (Fig. 4a,b), whereas the piezoresponse amplitude (Fig. 4c,d) disappears with decreased driving voltage, consistent with a piezoelectric effect. It should also be noted that a PFM image provides better resolution than topographic imaging and reveals more detail of the fibril structure.

The ability to map electromechanical properties in aqueous media opens the way to characterization of biological systems in native-like conditions. We propose to expand PFM for characterization and stimulation of live cells in a physiological environment. Our long-term vision is to use electromechanical imaging as a diagnostic tool (in the simplest example, electromechanical activity of a muscle cell differs depending on whether it is alive or not) and ultimately, to utilize electromechanical stimulation for induction of a desirable change in cell behavior (e.g., change of phenotype, apoptosis, or contraction). More specifically, we will focus on electromechanical properties of myocytes as a model system because of strong electromechanical coupling observed in these cells. Application of action potential results in the opening of membrane-associated ion channels. This allows the influx of Ca2+ ions, which triggers the contractile machinery.

It is expected that interaction of PFM tip with a voltage-dependent ion channel will induce its opening if the tip bias exceeds the action potential of the corresponding ion channel. This in turn will result in mechanical change detectable by PFM. The expected outcome of the experiment is high-resolution mapping of the distribution of ion channels on a myocyte surface. It will also be possible to determine action potentials of individual ion channel molecules because ion channel opening is impossible if the tip bias is less than the action potential. If two ion channels with different action potentials are present, it will be possible to map them separately.

Further, we plan to expand this project to electromechanical mapping of vascular smooth muscle cells (VSMCs). The shift from the contractile phenotype of the VSMCs to their synthetic phenotype is associated with vascular dysfunction after injury and plays a critical role in development of a number of vascular conditions, including atherosclerosis and restenosis. Studies of electromechanical properties of these two phenotypes and their response to electromechanical stimuli represent an ideal model system for understanding cell electromechanics. This project, which will develop experimental techniques necessary for understanding and predicting cell behavior upon electromechanical stimulation, ultimately will result in the design of novel diagnostic and therapeutic devices capable of detecting pathological changes in cells and using feedback control to provide the treatment of vascular diseases through electromechanical stimulation. Moreover, the developed experimental, methodological, and theoretical framework will be universally applicable to other types of cellular systems.This is an ongoing collaborative project with Dr. Sergei Kalinin (ORNL, PFM imaging) and Dr. Philip Rack (University of Tennessee, Knoxville, fabrication of shielded tips).

Scanning probe microscopy (SPM) allows imaging with nanoscale resolution in ambient conditions. Different variants of SPM that are sensitive to a number of physical properties, such as surface topography (AFM), conductivity (STM), electric charge (EFM), or magnetic properties (MFM), have been developed and used for measuring the distribution of the corresponding property on the nanoscale. Mapping a chemical property presents a much greater challenge and can only be achieved in some special cases using indirect methods. No general SPM technique has yet been developed to detect the presence of a specific chemical or biochemical structure. Availability of such technique would have a tremendous impact on imaging, sensing, and manipulation of small objects, including single molecules and their complexes, interfaces, and individual defects in crystals. We explore the possibilities to use liquid AFM coupled with Förster Resonant Energy Transfer (FRET) or metal-induced fluorescence quenching to achieve mapping of the distribution of biomolecules of interest on the surface of live cells.