Engineered Bacteriophage Enabled Nanoprobes for SKBR-3 Cancer Cell Specific Imaging and Therapy

Abstract

With the recent progress in nanoscience and great deal of understanding in molecular biology, it is now possible to combine genetic tools with synthetic nano- constructs for improved biotechnology applications. Viruses with its inherent nanosize architecture, genetic flexibility, stability to harsh conditions, circulatory behavior and targeting ability, in combination with nano science, are currently an excellent source for designing nano based therapeutics for cancer diagnosis and treatment.

Here, we integrate phage display technology (PDT) with nanotechnology to synthesize novel nano-conjguates for potential biomedical applications.

The first part of this dissertation is focused on the identification and characterization of novel SKBR-3 breast cancer cell targeting/internalizing ligands using landscape phage libraries. We used several computational methods and biochemical approaches to characterize the specificity, affinity and selectivity of the screened bacteriophage against target SKBR-3 breast cancer cells. In order to understand the mechanism of entry, we investigated the actin dynamics by using live cell and fluorescence imaging during selected phage internalization into target SKBR-3 cells. In conclusion, we demonstrate that phage harboring VSSTQDFP and DGSIPWST peptides could selectively internalize into SKBR-3 cells with high affinity and show rapid involvement of membrane ruffling and rearrangements of actin cytoskeleton during phage entry.

The second part of this dissertation is focused on the isolation of major coat proteins, pVIII from screened bacteriophage, its conjugation on functionalized gold nanorod using appropriate chemistry and its multipurpose applications. The successful conjugation of coat proteins on functionalized gold nanorods was verified by using spectroscopic and microscopic techniques. In conclusion, we demonstrate that the resulting protein/peptide- nanoconjugates can be used for imaging and selective photo thermal destruction of SKBR-3 breast cancer cells upon exposure to near-infrared (NIR) light. In order, to gain insight into the mechanism and understand the key cellular processes involved following the treatment of these nanoconjugates, we used illumina microarray technique to explore the molecular interactions with in our model SKBR-3 breast cancer cells. We identified 76 genes to be up regulated and 26 genes are down regulated following the treatment of protein nanoconjugates. Our recent preliminary animal studies in SKBR-3 tumor model (nude mice) shows that these protein-nanoconjugates are feasible for in vivo applications. However, more in vivo experiments are under investigations before its clinical significance is realized.

The third part of this dissertation is focused on the assembly of coat proteins on functionalized carbon nanotubes and other biomedical applications of engineered bacteriophage in nanoscience. Using various spectroscopic and microscopic studies, we demonstrate phage proteins assembled on carbon nanotubes can be used for imaging purpose, and SKBR-3 specific bacteriophage can be used as a template for conjugation of photosensitizer, pyropheophorbid-a for targeted photo dynamic therapy. We also demonstrate filamentous bacteriophage displayed with eight glutamic acid residues [E8] on the N-terminus of major coat protein using phage display can be used as a template to assemble liposomes and form phage-liposome complex for targeted drug delivery.