E-Book Overview
Expert laboratorians update the classic bioconjugation methods and introduce valuable new techniques that go beyond pure biomedical applications to include elements from advanced organic synthesis, molecular biology, and materials science. These readily reproducible methods cover the preparation of protein conjugates using covalent and noncovalent conjugation, the synthesis of nucleic acid conjugates using a variety of labeling techniques, and approaches to semisynthetic conjugates of proteins. Additional chapters address the biofunctionalization of inorganic surfaces, including the on-chip synthesis of peptide nucleic acids to generate microarrays for the high-throughput analysis of RNA and DNA, gold nanaoparticles, and carbon nanotube probes for atomic force microscopy.
E-Book Content
Catalase–Antibody Nanoconjugates
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1 Streptavidin–Biotin Crosslinking of Therapeutic Enzymes With Carrier Antibodies Nanoconjugates for Protection Against Endothelial Oxidative Stress Vladimir V. Shuvaev, Thomas Dziubla, Rainer Wiewrodt, and Vladimir R. Muzykantov
Summary The streptavidin–biotin system may be used to synthesize immunoconjugates for targeted delivery of drugs, including therapeutic enzymes. The size of antibody–enzyme conjugates, which is controlled by the extent of biotinylation and molar ratio between the conjugate components, represents an important parameter that in some cases dictates subcellular addressing of drugs. This chapter describes the methodology of formation and characterization of polymeric immunoconjugates in the nanoscale range. A theoretical model of streptavidin conjugation based on general principles of polymer chemistry is considered. Factors that influence size and functional characterization of resulting polymer conjugates, as well as advantages and limitations of this approach, are described in detail. The protocols describe the formation of immunoconjugates possessing an antioxidant enzyme, catalase, directed to endothelial cells by anti-platelet endothelial cell adhesion molecule antibodies. However because of the modular nature of the streptavidin–biotin crosslinker system, the techniques herein can be easily adapted for the preparation of nanoscale immunoconjugates delivering other protein drugs to diverse cellular antigens. Key Words: Immunoconjugates; vascular immunotargeting; polymerization; nanoscale carrier; catalase; streptavidin; biotin; dynamic light scattering; drug delivery.
1. Introduction Targeted drug delivery, as attained by conjugating therapeutic enzymes with affinity carrier antibodies, promises a significant improvement over the current therapeutic means and, therefore, has remained the focus of intense research From: Methods in Molecular Biology, vol. 283: Bioconjugation Protocols: Strategies and Methods Edited by: C. M. Niemeyer © Humana Press Inc., Totowa, NJ
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for several decades. For example, endothelial cells lining the luminal surface of the vasculature represent an important target for delivery of antithrombotic, anti-inflammatory, antioxidant agents and genetic materials. Cell adhesion molecules (e.g., platelet endothelial cell adhesion molecule [PECAM] and intercellular adhesion molecule [ICAM]) represent very attractive endothelial determinants for vascular immunotargeting, for example, in the context of inflammation. Some drugs require intracellular uptake. Recent studies revealed that although endothelial cells do not internalize monomeric antibodies against PECAM and ICAM, one can facilitate intracellular delivery of therapeutic cargoes by controlling size of the anti-PECAM and anti-ICAM immunoconjugates in the nanoscale range (1–3). The biotin–streptavidin system can be used to synthesize nanoscale therapeutic immunoconjugates, providing an interesting alternative to other commonly pu