Stealth Carbon Nanotubes: Strategies to Coat Carbon Nanotubes to Prevent Opsonization and Improve Biodistribution

Carbon nanotubes (CNT) have recently been in the limelight for its potential role in disease diagnostics and therapeutics. Even before these medical applications can be realized, there is a need to address issues like opsonization, phagocytosis by macrophages and sequestration to liver and spleen for eventual elimination from the body. We believe coating CNT with biocompatible and opsonin resistant moieties will not only help CNT in traversing the blood stream to reach the target organ, but also improve biodistribution tremendously. We set out to achieve this by firstly identifying a new compound, mAmp, which is a fluorescent derivative of the antibiotic, Ampicillin. Besides possessing a varied plethora of properties that complements CNTs already superlative traits, mAmp also affords opsonin resistance to CNT. We wanted to test and compare the four categories of opsonin repellants that we employed: synthetic - Polyethylene glycol (PEG), semisynthetic - mAmp, seminatural - Dextran sulfate (DSS) and natural - Protein A (PrA) + Factor H (FH). We developed novel strategies to conjugate these moieties to CNT and in the process also implemented attachment of Antibodies, specific recognition moieties, to these hybrids to make them ready for precision targeting downstream. Of the four materials used, DSS posed considerable difficulties in achieving a pure DSS-CNT hybrid mainly due to its size and lack of defined purification strategies to separate polysaccharides in a mixture. We responded to this challenge by devising a simple lectin based affinity chromatography system that employs CNT as the support material. As proof of principle we tested the four hybrids on Staphylococcus aureus, in their ability to evade the bacterium in the absence of specific antibody and ability to specifically attach to the bacterium in its presence. We then tested the particles on human macrophages in the presence of opsonins, C3b and IgG. It was henceforth proved that coating CNT with the opsonin resistant moieties provided excellent immunity versus macrophages and considerable stealth character to CNTs

DNA-Directed Self-Assembly of Microscopic 1-D Carbon Nanotube Wire

Molecular wire construction using DNA-directed selfassembly of multi-walled carbon nanotubes (MWNTs) has been attempted. MWNTs were selected owing to their exceptional electrical and structural properties. DNA, which possesses specific molecular recognition properties, served as the engine for the assembly. Non-crosshybridizing (NCH) sequences were designed to impart specificity and high throughput reaction. Stepwise manufacturing of the wire was accomplished by first functionalizing the NCH 20 base pair single-stranded sequences to the tips of MWNT using amide linkage. The adducts were then utilized to self-assemble 1-D nanotube wire through DNA hybridization reactions between two complementary ssDNAs functionalized to the MWNT tips. TEM, epi-fluorescent microscopy and AFM analyses showed successful assemblies of micrometer-scale 1-D MWNT-DNA wires, ranging from 2.7-20 μm. The results demonstrate great potentials of the DNA-guided self-assembly process, which would provide an uncomplicated, versatile and inexpensive way to manufacture micrometer-scale molecular wires. © 2007 IEEE

In situ fluorescence microscopy visualization and characterization of nanometer-scale carbon nanotubes labeled with 1-pyrenebutanoic acid, succinimidyl ester

In order to characterize hybrid bio/abio technology utilizing carbon nanotubes (CNTs), in situ, real-time, yet noninvasive methods of accurate and reliable imaging are needed for observing CNTs\u27 interactions with biological materials, i.e., DNA, in biologically relevant aqueous environments. Optical visualization and characterization of individual CNTs in aqueous solutions were explored in this study using 1-pyrenebutanoic acid, succinimidyl ester (PSE) and a conventional fluorescence microscope. The results demonstrate the potential of fluorescence microscopy based on PSE-based staining methodology monitoring with nanometer resolution of individual CNTs and their manipulation with biological materials in bio/abio hybrid systems. © 2006 American Institute of Physics

Antibody Quantum Dot Conjugates Developed via Copper-Free Click Chemistry for Rapid Analysis of Biological Samples Using a Microfluidic Microsphere Array System

Antibody-based proteomics is an enabling technology that has significant implications for cancer biomarker discovery, diagnostic screening, prognostic and pharmacodynamic evaluation of disease state, and targeted therapeutics. Quantum dot based fluoro-immunoconjugates possess promising features toward realization of this goal such as high photostability, brightness, and multispectral tunability. However, current strategies to generate such conjugates are riddled with complications such as improper orientation of antigen binding sites of the antibody, aggregation, and stability issues. We report a facile yet effective strategy to conjugate anti-epidermal growth factor receptor (EGFR) antibody to quantum dots using copper-free click reaction, and compared them to similar constructs prepared using traditional strategies such as succinimidyl-4-(<i>N</i>-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and biotin–streptavidin schemes. The F_c and F_ab regions of the conjugates retain their binding potential, compared to those generated through the traditional schemes. We further applied the conjugates in testing a novel microsphere array device designed to carry out sensitive detection of cancer biomarkers through fluoroimmunoassays. Using purified EGFR, we determined the limit of detection of the microscopy centric system to be 12.5 ng/mL. The biological assay, in silico, was successfully tested and validated by using tumor cell lysates, as well as human serum from breast cancer patients, and the results were compared to normal serum. A pattern consistent with established clinical data was observed, which further validates the effectiveness of the developed conjugates and its successful implementation both in vitro as well as in silico fluoroimmunoassays. The results suggest the potential development of a high throughput in silico paradigm for predicting the class of patient cancer based on EGFR expression levels relative to normal reference levels in blood

Radionuclides transform chemotherapeutics into phototherapeutics for precise treatment of disseminated cancer

Most of the systemic cancer therapies lack spatiotemporal control. Here, the authors show targeted activation of a light-sensitive drug by radiopharmaceuticals in disseminated cancer cells as potential in vivo treatment of metastatic diseases with reduced off-target toxicity