Vladimir Reukov

Proteins conjugated to poly(butyl cyanoacrylate) nanoparticles as potential neuroprotective agents

Poly(butyl cyanoacrylate) (PBCA) nanoparticles (NPs) can penetrate blood–brain barrier providing the means for drug delivery to the central nervous system. Here, we study attachment of superoxide dismutase (SOD) and anti‐glutamate N‐methyl D‐aspartate receptor 1 (NR1) antibody to PBCA NPs with the ultimate goal to design neuroprotective therapeutics for treatment of secondary spinal cord injury. Synthesis of monodispersed, ∼200 nm‐diameter PBCA NPs was performed using polymerization at pH 2.0 with Dextran 70,000 as the stabilizer. Sulfo‐HSAB spacers were used to covalently attach SOD and NR1 antibodies to the dextran‐coated NPs. The prepared protein–NP conjugates possessed SOD activity and were capable of binding to rat cerebellar neurons. Thus, SOD and NR1 antibodies may be simultaneously attached to PBCA NPs while retaining at least a fraction of enzymatic activity and receptor‐binding ability. The conjugates showed neuroprotective efficacy in vitro with rat cerebellar cell cultures challenged by superoxide. Biotechnol. Bioeng. 2011;108: 243–252.

Double-Layer Mediated Electromechanical Response of Amyloid Fibrils in Liquid Environment

Harnessing electrical bias-induced mechanical motion on the nanometer and molecular scale is a critical step toward understanding the fundamental mechanisms of redox processes and implementation of molecular electromechanical machines. Probing these phenomena in biomolecular systems requires electromechanical measurements be performed in liquid environments. Here we demonstrate the use of band excitation piezoresponse force microscopy for probing electromechanical coupling in amyloid fibrils. The approaches for separating the elastic and electromechanical contributions based on functional fits and multivariate statistical analysis are presented. We demonstrate that in the bulk of the fibril the electromechanical response is dominated by double-layer effects (consistent with shear piezoelectricity of biomolecules), while a number of electromechanically active hot spots possibly related to structural defects are observed.

Protein?nanoparticle conjugates as potential therapeutic agents for the treatment of hyperlipidemia

Hyperlipidemia, a condition associated with atherosclerosis, can develop because of the lack of low density lipoprotein (LDL) receptors in hepatocytes. Since injected polymeric nanoparticles are quickly taken up by the liver Kupffer cells, we hypothesize that it is possible to enhance LDL delivery to the liver through the use of LDL-absorbing nanoparticles. Here, we demonstrate the feasibility of the proposed approach in vitro. We used biodegradable and biocompatible polylactide nanoparticles (approximately 100 nm in diameter) with covalently attached apolipoprotein B100 antibody to adsorb LDLs at physiologically relevant concentrations. We showed that up to sixfold decreases of LDL levels can be achieved in vitro upon treatment of LDL suspensions (500 mg dl( - 1)) with anti-apoB100-nanoparticle conjugates. The study of the uptake of the antibody-nanoparticle-LDL complexes by cells was performed using a mouse macrophage cell line (RAW 264.7) as a model for liver Kupffer cells. We found that macrophages can quickly take up antibody-nanoparticle-LDL complexes and digest them within 24 h. No evidence of cytotoxicity was observed for the experimental conditions used in this study.

Functional recognition imaging using artificial neural networks: applications to rapid cellular identification via broadband electromechanical response

Functional recognition imaging in scanning probe microscopy (SPM) using artificial neural network identification is demonstrated. This approach utilizes statistical analysis of complex SPM responses at a single spatial location to identify the target behavior, which is reminiscent of associative thinking in the human brain, obviating the need for analytical models. We demonstrate, as an example of recognition imaging, rapid identification of cellular organisms using the difference in electromechanical activity over a broad frequency range. Single-pixel identification of model Micrococcus lysodeikticus and Pseudomonas fluorescens bacteria is achieved, demonstrating the viability of the method.

Targeted delivery of therapeutic enzymes

Enzymes have great potential as therapeutic agents due to their high activity and selectivity. However, broader use of enzyme drugs in clinical practice is limited due to their poor stability, immunogenicity and potential systemic toxicity. Targeted delivery of therapeutic enzymes can in many cases provide solutions to these problems. Here we review recently developed strategies for targeted delivery of several most important classes of enzyme drugs and discuss their advantages and potential limitations. While a variety of approaches have been proposed and studied in vitro and in animal models, further investigations and clinical trials are needed to make these delivery systems suitable for clinical applications.

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.

Antioxidant Activity of SOD and Catalase Conjugated with Nanocrystalline Ceria

Interactions of nanoparticles with biological matter—both somatically and in nature—draw scientists’ attention. Nanoparticulate systems are believed to be our saviors, acting as versatile drug delivery vehicles. However, they can also cause life-threatening bodily damage. One of the most important properties of nanocrystalline cerium dioxide is its antioxidant activity, which decreases the abundance of reactive oxygen species during inflammation. In this paper, we report on synergistic effects of inorganic cerium oxide (IV) nanoparticles conjugated with the antioxidative enzymes superoxide dismutase and catalase on scavenging oxygen and nitrogen radicals.

Relationship between Targeting Efficacy of Liposomes and the Dosage of Targeting Antibody Using Surface Plasmon Resonance.

Surface plasmon resonance (SPR) was used in this research to investigate the targeting efficacy (i.e., the binding affinity) of antibody-modified liposomes. The results indicated that liposomes modified by targeting antibodies exhibited an increase in apparent binding affinity, a result attributed to the avidity effect. More specifically, the targeting effect improved as the surface density of the targeting antibody increased, an increase primarily attributed to the decrease of the dissociation rate. However, this trend stopped when the surface density reached a threshold of approximately 1.5 × 10(8) antibody/mm(2). This surface density was found to be quite consistent regardless of the liposome size and the type of targeting antibody. In addition, a traditional cell binding experiment was conducted to confirm the saturation point obtained from SPR.

Electromechanical and elastic probing of bacteria in a cell culture medium

Rapid phenotype characterization and identification of cultured cells, which is needed for progress in tissue engineering and drug testing, requires an experimental technique that measures physical properties of cells with sub-micron resolution. Recently, band excitation piezoresponse force microscopy (BEPFM) has been proven useful for recognition and imaging of bacteria of different types in pure water. Here, the BEPFM method is performed for the first time on physiologically relevant electrolyte media, such as Dulbeccos phosphate-buffered saline (DPBS) and Dulbeccos modified Eagles medium (DMEM). Distinct electromechanical responses for Micrococcus lysodeikticus (Gram-positive) and Pseudomonas fluorescens (Gram-negative) bacteria in DPBS are demonstrated. The results suggest that mechanical properties of the outer surface coating each bacterium, as well as the electrical double layer around them, are responsible for the BEPFM image formation mechanism in electrolyte media.

Anti-inflammatory coatings of hernia repair meshes: A pilot study

The current prevalence of postoperative chronic pain from hernioplasty procedures employing polymer mesh is close to 30%. Most of the researchers agree that oxidative stress, resulting from the release of oxidants and enzymes during acute inflammatory response, is a key factor in the development of posthernioplasty complications. This results in both the decrease of the biomechanical properties and stiffening of the polymer fibers of the mesh, leading to chronic pain. Moreover, enhanced activity of inflammatory cells can lead to an excessive deposition of connective tissue around the implant. In this study polypropylene hernia repair meshes coated with vitamin E (α-tocopherol), a known antioxidant, were prepared and characterized. The absorption isotherm of vitamin E on the mesh was characterized and a release profile study yielded a promising results, showing sustained release of the drug over a 10-day period. An animal study was conducted, and histological analysis five weeks after implantation exhibited a reduced host tissue response for a modified mesh as compared to a plain mesh, as evidenced by a higher mature collagen to immature collagen ratio, as well as lower level of fatty infiltrates, neovascularization and fibrosis in the case of modified mesh. These results support the use of α-tocopherol as a potential coating in attempt to reduce the extent of postoperative inflammation, and thereby improve long-term outcomes of hernioplasty.