PhD Thesis Defense Seminar
Tuesday, July 14, 2015
K-307, Med 3-6408
“Discovery and Characterization of Antibodies that Bind Nanoparticles”
Presented by: Supriya Ravichandran
Supervised by: Prof. Lisa DeLouise
Nanoparticle (NP) safety concerns stem from their unique physiochemical properties such as high surface area to volume ratio and small size, and reactivity otherwise not present in the bulk form. These NP properties contribute to the potential toxicity and altered tissue function when in contact with biological systems. Since skin is one of the major routes of NP entry into the system upon contact with NP-enabled products, researchers have focused on determining if NPs can penetrate the stratum corneum, which is the outermost skin barrier layer. Semiconductor quantum dots (QDs) and metal oxide NPs (titanium dioxide (TiO2)) have been widely used to study NP-skin interactions due to their commercial importance. However, studies show varying results on NP skin penetration depending upon the NP size and surface chemistry, skin model used and the NP detection techniques employed. Conventional techniques employed to detect NPs in tissues such as transmission electron microscopy coupled with energy dispersive x-ray spectroscopy offer superior nanoscale resolution, however pose limitations due to the high cost of sample processing and limited sample analysis throughput. Confocal and fluorescence microscopy are also common techniques used to detect fluorescent NPs, however their detection ability is often obscured by tissue autofluorescence and are limited to detecting fluorescent NPs. Therefore, a simple economical technique which can provide information on both the presence of NPs and their form in biological systems and the environment is required.
We have developed NP binding antibodies to commercially important NPs including QDs and TiO2 NPs using phage display technology. Phage display is used to identify protein or peptide binders to a wide variety of targets. Typically, nucleotide sequences encoding the protein/peptide library are fused to a gene encoding a phage coat protein thus allowing them to be displayed on the phage exterior. An affinity based selection technique (biopanning) is used to identify binders from the library. In this work, we have developed antibodies to NPs from a phage library containing ~2x109 unique single-chain variable fragment (scFv) antibodies each displayed monovalently on the gene III coat protein of a M13 filamentous phage. The scFv antibodies are engineered with a FLAG tag to allow for secondary detection using standard immunohistochemistry methods. This thesis discusses the discovery of novel antibodies binding QDs and TiO2 NPs and their functionality by demonstrating their binding both in vitro and in an ex vivo human skin model. The antibodies isolated against GSH-QDs and TiO2 NPs by panning in solution, can recognize the respective NPs in skin and did not show any non-specific binding to skin samples without NPs. Non-fluorescent TiO2 NPs were detected using simple microscopic techniques with the scFv antibody isolated against them. The antibodies do not exhibit non-specific binding to dissimilar NPs such as gold NPs or carbon nanotubes as demonstrated through custom-designed in vitro assays. Additionally, the antibodies have been characterized for their binding and cross-reactivity properties to several other NPs, and some challenges associated with the isolation of the antibodies from a large library and alternative method for selection of antibodies have been discussed. It was found that enrichment on NPs in solution does not render off-target clones or false positives when compared to enrichment on immobilized target, conventionally used in phage display. The novel antibodies isolated when used in conjunction with other existing techniques for NP detection will comprise a powerful tool kit, and enable researchers to use them to detect NPs both in the environment and in a biological milieu.