Direct attachment of nanoparticle cargo to Salmonella typhimurium membranes designed for combination bacteriotherapy against tumors.

Nanoparticle technology is an emerging approach to resolve difficult-to-manage internal diseases. It is highly regarded, in particular, for medical use in treatment of cancer due to the innate ability of certain nanoparticles to accumulate in the porous environment of tumors and to be toxic to cancer cells. However, the therapeutic success of nanoparticles is limited by the technical difficulty of fully penetrating and thus attacking the tumor. Additionally, while nanoparticles possess seeming-specificity due to the unique physiological properties of tumors themselves, it is difficult to tailor the delivery of nanoparticles or drugs in other models, such as use in cardiac disease, to the specific target. Thus, a need for delivery systems that will accurately and precisely bring nanoparticles carrying drug payloads to their intended sites currently exists. Our solution to this engineering challenge is to load such nanoparticles onto a biological "mailman" (a novel, nontoxic, therapeutic strain of Salmonella typhimurium engineered to preferentially and precisely seek out, penetrate, and hinder prostate cancer cells as the biological delivery system) that will deliver the therapeutics to a target site. In this chapter, we describe two methods that establish proof-of-concept for our cargo loading and delivery system by attaching nanoparticles to the Salmonella membrane. The first method (Subheading 1.1) describes association of sucrose-conjugated gold nanoparticles to the surface of Salmonella bacteria. The second method (Subheading 1.2) biotinylates the native Salmonella membrane to attach streptavidin-conjugated fluorophores as example nanoparticle cargo, with an alternative method (expression of membrane bound biotin target sites using autodisplay plasmid vectors) that increases the concentration of biotin on the membrane surface for streptavidin-conjugated nanoparticle attachment. By directly attaching the fluorophores to our bacterial vector through biocompatible, covalent, and stable bonds, the coupling of bacterial and nanoparticle therapeutic approaches should synergistically lead to improved tumor destruction.