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Nano-Therapeutics: Bacterial Mediated Delivery of Nanoparticles into Cells

Demir Akin, Jennifer Sturgis^, Kristin Burholder*, Arun Bhunia*, J. P. Robinson^, S. Muhammad^, R. Bashir,

School of Electrical and Computer Engineering, Weldon School of Biomedical Engineering, ^Department of Basic Medical Sciences, *Department of Food Science, Purdue University

The recent advancements in nanotechnology have been utilized to introduce smart nano-particles into mammalian cells for delivery of genes and proteins in hopes to monitor or intervene signal transduction pathways. In addition, the ability of bacteria to mediate gene transfer into mammalian cells, a method termed ‘bactofaction’, has also been recently demonstrated as a method for non-viral gene therapy. In this project, we demonstrate the integration of these two technologies to develop a simple yet powerful method to deliver genes loaded on nanoparticles which in turn are carried on the surface of bacteria. As an in-vitro proof of concept demonstration for delivery of genetic material, we anchored nano-particles onto surface of bacteria and then functionalized the nanoparticles with green fluorescence protein (GFP) reporter plasmids. Bacteria entered the cytosol of the target cells and delivered their payloads into the cytosol, escaping from the sub-cellular vesicles possibly by the pore forming action of listeriolysin-O (LLO). The transgene, disassociated from the nanoparticle and bacteria and were targeted to the nucleus of the cells resulting in the expression of GFP signal in the cytoplasm. We examined the delivery of the nano-particles inside cells and subsequent expression using fluorescence and confocal microscopy and flow cytometry. The demonstrated approach, applied with attenuated strains of bacteria, may be used to actively deliver multiple particles of different sizes, each loaded with different therapeutic or imaging agent into a variety of cells.

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