(599q) Characterization of Nanoscale Bacteria Enabled Autonomous Drug Delivery Systems (NanoBEADS) Transport in an in-Vitro Tumor Model

The lack of efficacy of existing chemotherapeutic treatments of solid tumors is partially attributed to the limited diffusion distance of drug particles which prevents the successful treatment of quiescent tumor cells. The low selectivity of anti-cancer drugs with respect to cancerous tissue is also problematic due to their exposure to healthy cells. Thus, chemotherapy can be enhanced through better drug carrier targeting towards cancerous cells. Several strains of attenuated bacteria, such as Escherichia coli and Salmonella Typhimurium have been identified to possess the natural ability to preferentially colonize tumor tissues. In this work, we demonstrate that tumor colonizing bacteria (E. coli and S. Typhimurium) can be coupled with therapeutic nanoparticles to form intelligent Nanoscale Bacteria Enabled Autonomous Drug Delivery Systems (Hereafter referred to as NanoBEADS). NanoBEADS are able to preferentially colonize tumors and therefore transport drug nanoparticles deep into the tumor.  We have demonstrated that NanoBEADS can penetrate deeper within the tumor tissue compared to passively diffusing chemotherapeutic nanoparticles; thus, they have the potential to dramatically improve the delivery of chemotherapeutic to solid tumors. Tumor spheroids were grown in-vitro using human colon carcinoma cells (HT-29) in low adhesion round bottom 96-well plates. For the construction of the NanoBEADS, GFP expressing E. coli MG1655 bacteria were grown in L-broth, then incubated with goat polyclonal anti-Lipid A LPS antibody labeled with biotin (10 µg/mL) for 1 hour at room temperature. Streptavidin coated 150-400 nm diameter particles were then mixed with the bacteria and incubated at room temperature for 30 minutes. For extravascular transport quantification, NanoBEADS suspended in McCoy’s 5a culture media were incubated with the tumor spheroids for 30 minutes. Tumors were then fixed in 4% paraformaldehyde for a period of 24 hours, rinsed in PBS and sectioned with a cryotome into 50 µm thick slices. Sections were imaged using fluorescence microscopy. Tumor sections were divided into two specific regions: viable rim and necrotic core, to quantify the distribution and penetration of passively diffusing nanoparticles and NanoBEADS within tumors. The distribution of the NanoBEADS as well as passively diffusing nanoparticles was quantified by measuring the fluorescence intensity in each region of interest. Our preliminary results indicate that NanoBEADS have the capability to transport loads deeper within the tumor tissue (10 % of NanoBEADS have penetrated the tumor past the viable rim as opposed to only 1% for passively diffusing particles). Passively diffusing nanoparticles have limited penetration in the tumor spheroid as opposed to bacteria carrying nanoparticles that penetrate the tumor farther. This study demonstrates that NanoBEADS can be used as live autonomous drug delivery agents with enhanced selectivity and penetration of solid tumors. As illustrated in this work, bacteria carrying particles can penetrate a solid tumor past the viable rim into the hypoxic region of the tumor.